An in vivo analyte sensor and a method of manufacturing an in vivo analyte sensor

Abstract

The present disclosure refers to an in vivo analyte sensor, comprising: a substrate (20); a conductive layer (21) disposed over at least a portion of the substrate (20); a sensing layer (24) disposed formed on the conductive layer (21), wherein the sensin\g layer (24) forms a sensing area configured to generate one or more signals identifying a monitored analyte level after insertion of the analyte sensor and when maintained in fluid contact with interstitial fluid; and a membrane layer (25) formed at least on the sensing layer (24). The sensing layer (24) is formed with an area sensitivity of 0,2 to 1,5 nA / (mqx / dL) mm2 within the sensing area. Further, a method of manufacturing an in vivo analyte sensor is provided.

Description

[0001]P38942 An in vivo analyte sensor and a method of manufacturing an in vivo analyte sensor The present disclosure refers to an in vivo analyte sensor, and a method of manufacturing or producing an in vivo analyte sensor. In vivo analyte sensors are applied for detecting characteristic for an analyte, such as a level of analyte. For example, an in vivo analyte sensor may be applied for continuously monitoring a glucose level for a patient, such as diabetes patient. The glucose level is continuously mon- itored after insertion of the analyte sensor which is maintained in fluid contact with interstitial fluid. For sensing signals identifying a monitored analyte level, the analyte sensor is having a work- ing electrode comprising a sensing area formed by disposing a sensing layer together with additional functional layers on a substrate of the analyte sensor. The working electrode of the analyte sensor can have a plurality of separated sensing areas. Such type of analyte sensor may also be referred to multi spot working electrode. With respect to the sensing signals, the plurality of sensing areas enables tissue averaging. Effects due to local differences with regard to the analyte level may be reduced. Further, analyte sensors provided with a working electrode having only one single sensing area have been proposed. Such type of analyte sensor may also be referred to single spot working electrode. Tissue averaging is achieved by employing larger area working electrodes. Document WO 2021 / 180977 A1 refers to a method for the preparation of a working electrode on a sensor substrate. The method is comprising the following: a) providing at least one sensor substrate comprising at least a first side, the first side having at least one conductive trace; b) applying at least one layer of at least one sensing material onto the first side of the sensor substrate, wherein the sensing material covers at least a portion of the at least one conductive trace; 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 is at least partially removed and wherein at least a second portion of the sensing material covering the at least one conductive trace is preserved on the first side of the sensor substrate to obtain at least one working electrode on the sensor substrate; and d) applying at least one membrane layer, the membrane layer at least partially covering the working electrode, wherein the membrane layer comprises at least P38942 one cross-linker for cross-linking at least a part of the sensing material. The method further comprises e) at least one diffusion step, wherein in the diffusion step the cross-linker com- prised in the membrane layer at least partially diffuses into the sensing material. Document EP 3714788 A1 discloses a method of manufacturing a batch of in vivo analyte sensor systems, comprising forming multiple in vivo analyte sensor systems, each in vivo an- alyte sensor system comprising an in vivo analyte sensor. The in vivo analyte sensor is having: a substrate; a conductive layer disposed over at least a portion of the substrate; a dielectric layer disposed over the conductive layer and having a void therein; a sensing layer disposed within the void and formed on the conductive layer to define an active area of the analyte sensor that generates one or more signals corresponding to a monitored analyte level after insertion of the analyte sensor and when maintained in fluid contact with interstitial fluid; and a membrane layer formed on the sensing layer; and a sensor electronics assembly may be coupled with the analyte sensor. The sensor electronics assembly is comprising a memory, determining the thickness of the membrane layer of at least one of the batch of analyte sensors and at one or more points of the membrane layer; determining a calibration parameter based on the determined thickness of the membrane layer of the at least one of the batch of analyte sensors; and storing the determined calibration parameter in the memory of the sensor elec- tronics assembly of each of the batch of in vivo analyte sensor systems during manufacture of the analyte sensor system. Each in vivo analyte sensor system further comprises a processor configured to correlate the generated one or more signals with the monitored analyte level by applying the determined calibration parameter to the generated one or more signals. It is an object to provide an in vivo analyte sensor, and a method of manufacturing or producing an in vivo analyte sensor which enable flexible sensor adjustment for different application sce- narios or conditions. For solving the object, an in vivo analyte sensor according to claim 1, and a method of manu- facturing or producing an in vivo analyte sensor according to claim 14 are provided. Further aspects are disclosed in dependent claims. According to one aspect, an in vivo analyte sensor is provided, comprising: a substrate; a conductive layer disposed over at least a portion of the substrate; a sensing layer disposed on P38942 the conductive layer, wherein the sensing layer forms a sensing area configured to generate one or more signals identifying a monitored analyte level after insertion of the analyte sensor and when maintained in fluid contact with interstitial fluid; and a membrane layer formed atleast on the sensing layer. The sensing layer is formed with an area sensitivity of 0,2 to 1,5^^(^^ ) ^ మ within the sensing area.^^ ^According to another aspect, a method of manufacturing an in vivo analyte sensor is provided, the method comprising: providing a substrate; disposing a conductive layer over at least a portion of the substrate; disposing a sensing layer on the conductive layer to form a sensing area configured to generate one or more signals identifying a monitored analyte level after insertion of the analyte sensor and when maintained in fluid contact with interstitial fluid; and forming a membrane layer at least on the sensing layer. The forming of the sensing layer com- prises forming the sensing layer with an area sensitivity of 0,2 to 1,5^^(^^ ) ^ మwithin the sensing ^^ ^area. The technology proposed provides an in vivo analyte sensor having an area sensitivity within a range of sensitivity which allows for a variety of sensor application scenarios.In some embodiments, the sensing layer may be formed with an area sensitivity of 0,2 to 1,3^^within the sensing area, alternatively with an area sen ^^(^^ ) ^^మ sitivity of 0,2 to 1,0 (^^ మ^^ ^^ ) ^^within the sensing area, and, according to still another alternative, with an area sensitivity of 0,2 to 0,8^^(^^ ) మwithin the sensing area. ^^ ^^The substrate of the in vivo sensor provides for a mechanical backbone of the analyte sensor ensuring mechanical stability. It is a carrier material for other layers of the in vivo analyte sen- sor. In certain embodiments, the substrate of the in vivo sensor may be a PET foil. The conductive layer may be formed with electrically conductive tracks. The conductive layer may provide electrical contact to patch electronics, for example, through a conductive rubber material. Further, the conductive layer may be configured to ensure adhesion to substrate. In certain embodiments, the (electrically) conductive layer may be made of a carbon-based ma- terial. P38942 In some embodiments, a dielectric layer may disposed over the conductive layer. The dielectric layer may be formed with a high electrical insulation resistance. The dielectric layer may be formed to protect the conductive layer from external influences. The membrane layer is formed to control or limit analyte diffusion from the interstitial fluid to the sensing area. For example, such limitation of diffusion may prevent enzymatic reaction limitation. The sensing area may be formed with an area size of 0,1 to 1,5 mm2. In some embodiments, the sensing layer may be formed with an area size of 0,1 to 1,0 mm2, alternatively an area size of 0,1 to 0,7 mm2, and, according to still another alternative, an area size between more than 1 mm2and 1,5 mm2. The sensing area may be formed with a sensitivity of 0,05 to 0,3 In some embodiments, the sensing area may be formed with a sensitivity of 0,05 to 0,2 and alternatively with a sensitivity of 0,05 to 0,15 The sensing area may be formed as a single spot sensing area. In this embodiment, a single sensing spot sensor can be provided. In operation of the analyte sensor, such as glucose level monitoring, averaging over local tissue regions is enabled by the single spot sensing area. In an embodiment, the in vivo analyte sensor may be formed as a single spot analyte sensor. The sensing layer may form a glucose sensing area configured to generate one or more signals identifying a monitored glucose level. The in vivo glucose sensor may be operable for contin- uous glucose monitoring. With respect to such embodiment, signals identifying the monitored glucose level are continuously generated fur subsequent signal analysis. The analyte sensor may further comprise a working electrode, a counter electrode, and a ref- erence electrode, wherein the working electrode comprises the sensing area. The working electrode, the counter electrode, and the reference electrode may be formed on the conductive layer. Thus, the conductive layer may form a carrier for such electrodes, i.e. electrode material P38942 forming the electrodes. The substrate may form an electrical isolator between the conductive material of the working electrode, the counter electrode, and the reference electrode. The working electrode comprising the sensing area generates analyte level dependent sensor signal. A signal current generated by the working electrode in combination with the counter / reference electrode. In an embodiment, the analyte sensor is formed with a single working electrode, the single working electrode comprising the single sensing area (single spot analyte sensor). The analyte sensor may be formed as a two-electrode analyte sensor comprising a combined counter and reference electrode. The analyte sensor is having two electrodes: the working electrode, and the combined counter and reference electrode. The combined counter and ref- erence electrode is operable to provide a relative potential of the working electrode. It is oper- able to from an electron current sink for sensor current from the working electrode to combined counter and reference electrode. The combined counter and reference electrode may be formed as an Ag | AgCl containing electrode on top of the conductive layer. In such embodi- ment, the combined counter and reference electrode provides Ag+-Ions for a counter electrode reaction. In general, for the different embodiments, the area sensitivity proposed here for the sensing area allows for higher working electrode area. Specifically, for the two-electrode analyte sensor design, the combined counter and reference electrode is likely to be consumed over time, and the place or area on a sensor (in area and thickness) for the material is limited by its small geometry. The area sensitivity proposed can achieve high working electrode areas for tissue averaging in combination with long-lasting capacity and functionality of the combined counter and reference electrode. The analyte sensor may further comprise a protecting layer deposited over at least one of the counter electrode, and the reference electrode. In some embodiment, the protecting layer may be deposited over a combined counter and reference electrode. The protecting layer may be a polymer-based layer. In certain embodiments, the protecting layer may be formed to limit or minimize Ag+-Ion leaching. P38942 The protecting layer may be deposited over a bending portion of the substrate. The analyte sensor may be bent in the bending portion for application, for example, during insertion of the analyte sensor. Having deposited over the bending portion will limit or prevent delamination of the layers in the bending portion or area. The membrane layer may be made of a flux-limiting or diffusion-limiting polymer membrane material. In case of an in vivo glucose sensor, the flux-limiting polymer membrane may be formed to control or limit glucose diffusion from the interstitial fluid to the working electrode to prevent enzymatic reaction limitation. Different flux or diffusion control may be implemented by varying layer thickness of the membrane layer. Examples for flux-limiting polymer membrane material can be found, for example, in WO 2022 / 106502 A1. In such embodiment, the mem- brane layer may also be referred to as flux-limiting or diffusion-limiting membrane layer. The membrane layer made of the flux-limiting polymer membrane material may be operable to ensure a response of sensor signals to glucose level as linear as possible by limiting diffu- sion of glucose to working electrode. Further, the membrane layer made of the flux-limiting polymer membrane material may be formed to prevent leakage of enzyme into the body of a patient. In addition or as an alternative, the membrane layer made of the flux-limiting polymer membrane material may be formed to provide at least one of the following: (i) reducing leakage of any water-soluble component from the analyte sensor into the body of the patient; (ii) reduc- ing concentration of interference substances at the working electrode, and (iii) biocompatibility of the sensor. The sensing area may be located in a sensor tip portion. In certain embodiments, locating the sensing area in the sensor tip portion may be combined with the single spot sensing area (single working electrode). This will allow for forming an analyte sensor short in length, since the working electrode can be positioned at the sensor tip portion without considering process tolerances for the space between multiple discrete structures. Therefore, the single spot sens- ing area can be positioned in a desired position in the tissue during insertion. The substrate may be a planar substrate. This will allow for forming a planar sensing area. The sensing layer may be made of an enzymatic Os-hydrogel based sensing material. P38942 In an embodiment, in the method of manufacturing the in vivo analyte sensor at least one of the following layers may be formed by a continuous coating process: the conductive layer, the sensing layer, and the membrane layer. Such method can avoid need for batch processes for manufacturing the analyte sensor. Also, the dielectric layer may be formed by a continuous coating process, if such layer is provided in the analyte sensor. The term "substrate", as used herein, is synonymously used with the term "sensor substrate" and specifically may refer to any kind of material or combination of materials, which is suitable to form a polymer layer to carry the working electrode and described herein. In particular, a "sensor substrate" as understood herein may comprise electrically insulating material. The term "layer", as used herein, specifically may refer to an element of a layer setup of the analyte sensor. Specifically, the term "layer" may refer to an arbitrary covering of an arbitrary substrate, specifically of a flat or planar substrate. The layer may specifically have a lateral extension exceeding its thickness by at least a factor of 2, at least a factor of 5, at least a factor of 10, or even at least a factor of 20 or more. Specifically, the analyte sensor may have a layer setup. One or more of the layers of the analyte sensor may comprise sublayers. For example, a layer comprising the conductive material may comprise at least one further layer. The term "electrically insulating or dielectric layer", as used herein, specifically may refer to a material or combination of materials which prevent the transfer of electrical charges and which do not sustain a significant electrical current. Specifically, the at least one electrically insulating material may be or may comprise at least one insulating resin, such as insulating epoxy resins used in manufacturing electronic printed circuit boards; in particular it may comprise or be a thermoplastic material such as polycarbonate, polyester like polyethylene terephthalate (PET), polyvinyl chloride (PVC), polyurethane, polyether, polyamide, polyimide or a copolymer thereof, such as glycol modified polyethylene terephthalate, polyethylene naphthalate, polytet- rafluorethylene (PTFE) or alumina. The term "conductive layer", as used herein, specifically may refer to a conductive strip, layer, wire or other type of elongated electrical conductor. Preferably, there is a planar conductive layer. More specifically, the term "conductive material" may refer to a material, which is con- ductive and hence capable of sustaining an electrical current, for example the conductive ma- terial may comprise at least one material selected from the group consisting of: carbon; carbon paste; gold; copper; silver; nickel; platinum; palladium. Specifically, the conductive material P38942 may be or may 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 material may be or may comprise at least one conductive compound, such as at least one conductive organic or inorganic compound. Additionally or alternatively, the at least one conductive material may be or may comprise at least one nonmetallic conductive material, e.g. polyaniline, poly-3,4- eth-ylenedioxythiophene (PEDOT), carbon or carbon paste. Carbon paste specifically may re- late to a material comprising carbon, a solvent such as diethyleneglycol butyl ether, and at least a binder such as vinyl chloride co- and terpolymers. Preferably, the conductive material may comprise gold and / or carbon; more preferably, the conductive material may consist of gold and / or carbon and / or carbon paste. Specifically the conductive material may comprise gold and a further material, for example carbon. Moreover, the conductive layer made of the conductive material may comprise at least one further layer of at least one further material; specifically the further layer may comprise a further conductive material. More specifically the further layer of the conductive material may comprise or may consist of carbon. The further material may be disposed on the first side. Using a further layer, in particular carbon, may contribute to efficient electron transfer by the conductive mate- rial. In some embodiments, an in vivo analyte sensor may be provided, comprising: a substrate, a conductive layer disposed over at least a portion of the substrate, a sensing layer disposed on the conductive layer, wherein the sensing layer forms a sensing area configured to generate one or more signals identifying a monitored analyte level after insertion of the analyte sensor and when maintained in fluid contact with interstitial fluid, and a membrane layer formed at least on the sensing layer, wherein the sensing area is having an area size of 0,1 to 1,5 mm2. In some embodiments, the sensing layer may be formed with an area size of 0,1 to 1,0 mm2, alternatively an area size of 0,1 to 0,7 mm2, and, according to still another alternative, an area size between more than 1 mm2and 1,5 mm2. Further, in some embodiments, a method of manufacturing an in vivo analyte sensor may be provided, the method comprising: providing a substrate, disposing a conductive layer over at least a portion of the substrate, disposing a sensing layer on the conductive layer to form a sensing area configured to generate one or more signals identifying a monitored analyte level after insertion of the analyte sensor and when maintained in fluid contact with interstitial fluid, P38942 and forming a membrane layer at least on the sensing layer, wherein the forming of the sensing layer comprises forming the sensing area with an area size of 0,1 to 1,5 mm2. With respect to the method of manufacturing the in vivo analyte sensor, the aspects or embod- iments disclosed above may apply mutatis mutandis. of embodiments Following further embodiments are described with reference to figures. In the figures show: Fig.1 a schematic representation of an in vivo analyte sensor; Fig.2A and 2B a schematic representation of a cross section of a portion of the analyte sen- sor; Fig.3 a schematic representation of embodiments of a tip portion of an in vivo an- alyte sensor; Fig.4 a graphical representation of normalized area sensitivity for an in vivo analyte sensor in dependence on an amount of PEG-DGE (poly(ethylene glycol) di- glycidyl ether) in a membrane layer of the analyte sensor; Fig.5 a graphical representation of normalized area sensitivity for an in vivo analyte sensor in dependence on a thickness of the membrane layer of the analyte sensor; and Fig.6 a graphical representation of normalized area sensitivity for an in vivo analyte sensor in dependence on a thickness of a hydrogel-based working electrode of the analyte sensor. Fig.1 shows a schematic representation of an in vivo analyte sensor 10. The analyte sensor 10 comprises a working electrode 11 having a sensing area 12, and a combined counter and reference electrode 13, thereby, forming a two-electrode analyte sensor. The sensing area 12 is configured to generate one or more signals identifying a monitored analyte level after inser- tion of the analyte sensor and when maintained in fluid contact with interstitial fluid. In an al- ternative embodiment, there can be a three-electrode analyte sensor comprising a counter electrode formed separately from a reference electrode. For the in vivo analyte sensor 10 dif- ferent positioning may be provided for the electrodes such as the working electrode 11 and the reference electrode 13. For example, in some embodiment, the working electrode 11 and the reference electrode 13 may be placed on opposite sides of the analyte sensor 10. P38942 For the embodiment shown, the working electrode 11 is formed as a single working electrode having the single sensing area 12 (single spot working electrode). The working electrode 11 is formed in a tip portion 14 of the analyte sensor 10. Both the working electrode 11, and the combined counter and reference electrode 13 are elec- trically connected to a conductive trace 15a, 15b which provide for electrical connection to contact pads for connecting electrical circuity for operation of the analyte sensor 10 (not shown). Fig.2A shows a schematic representation of a cross section of a portion of the analyte sensor 10 with the working electrode 11. On a substrate 20 being a planar substrate a conductive layer 21 is deposited. Over the con- ductive layer 21 an optional dielectric or electrically insulating layer 22 is deposited. In some other embodiment, the dielectric layer 22 may be omitted. In the embodiment shown, the die- lectric layer 22 is formed with a void 23 in some alternative embodiment. Within the void 23 a sensing layer 24 made of at least one sensing material is deposited on the conductive layer 21 to form the sensing area 12 configured to generate one or more signals identifying a moni- tored analyte level after insertion of the analyte sensor 10 and when maintained in fluid contact with interstitial fluid. The sensing layer 24 made of the sensing material forming the sensing area 12 is present within the working electrode 11 only. In the embodiment shown, a membrane layer 25 is formed for covering. In some alternative embodiment (see Fig. 2B), the analyte sensor 10 is free of the void 23. Also, the analyte sensor 10 is free of the dielectric layer 22. Rather, the sensing layer 24 made of at least one sensing material is deposited on the conductive layer 21 to form the sensing area 12 without forming the void 23 before. Fig. 3 shows different embodiments of a tip portion of the analyte sensor 10 based on the embodiment in Fig.2B. The sensing layer 24 forming the sensing area 12 of the working elec- trode 13 is deposited on the conductive layer 21 which is covered by the electrically insulating layer 22 in part (see left hand side). In the embodiment shown, a combined counter and refer- ence electrode 26 is formed (see right hand side). P38942 The analyte sensor 10 may be an in vivo analyte sensor suitable for at least paristial implan- tation into a body tissue of a user, more specifically an analyte sensor for continuous monitoring of the analyte. The analyte sensor 10 is an electrochemical sensor comprising working elec- trode 11, and the combined counter and reference electrode 13, and respective circuitry (not shown). More particularly, the analyte sensor 10 is an amperometrie electrochemical sensor comprising the working electrode 11. Typically, the analyte sensor 10 comprises at least one further electrode, particularly the combined counter and reference electrode 13. The working electrode 10 may be sensitive for the analyte to be measured at a polarization voltage which may be applied between the working electrode 10 and the combined counter and reference electrode 13 and which may be regulated by a potentiostat. A measurement signal may be provided as an electric current between the working electrode 10 and the com- bined counter and reference electrode 13. In such embodiment, a separate counter electrode is absent, and a pseudo or combined reference electrode is present, which is working as a counter electrode as well. Thus, the analyte sensor 10 may comprise a set of two or a set of three electrodes. Following, an embodiment for preparing the analyte sensor 10 is described. Additional meth- ods of preparation are known as such, for example, from WO 2022 / 106502 A1. The substrate 20 is based on polyethylene terephthalate. Suitable Carbon conductive inks are available from Henkel AG & Co. KGaA, Ercon, Inc. (Wareham, MA), E.I. du Pont de Nemours and Co. (Wil- mington, DE), Emca-Remex Products (Montgomeryville, PA), or TEKRA, A Division of EIS, Inc (New Berlin, Wl). Afterwards, the carbon paste was dried. The sensing material forming the sensing layer 22 was applied on the substrate 20 by cannula- coating and subsequently dried at ambient temperature, e.g. about 25 °C. The sensing material comprised 57% by weight of a polymeric transition metal complex (modified poly(vinylpyridine) backbone loaded with poly(biimidizyl) Os complexes covalently coupled through a bidentate linkage), 33 % by weight of glucose dehydrogenase and 10 % by weight of PEG-DGE (poly- ethylene glycol)-diglycidylether) in each case based on the sum of the percentages by weight of the polymeric transition metal complex, glucose dehydrogenase and PEG-DGE. By means of laser ablation, the working electrode 11 was formed. The analyte sensor 10 was prepared with different layer thickness of hydrogel-based coating (see Fig.6 below). P38942 The working electrode 11 of each sensor was coated (dip coating) with a different composition comprising a poly(vinylpyridine)-based polymer (see, for example, WO 2022 / 106502 A1) and a crosslinker PEG-DGE. Thereby, the membrane layer 25 was made of a flux-limiting or diffu- sion-limiting polymer membrane material. In such embodiment, the membrane layer 25 may also be referred to as flux-limiting or diffusion-limiting membrane layer (DLM). After coating the analyte sensor was dried and the polymer crosslinked at room temperature to obtain a flux-limiting membrane, specifically on the working electrode 11. Silver / silver chlo- ride was used for forming the combined counter reference electrode 13. The analyte sensor was prepared with different layer thickness for DLM (see Fig.5 below) and different composition with PEG-DGE linker (see Fig.4 below). In all cases, a solvent mixture of 80 % (v / v) ethanol and 20 % (v / v) water was used for the dip-coating process. Different examples for the analyte sensor 11 were investigated. An area sensor sensitivity SAfor the sensing area 12 was tuned. Specifically, different glucose levels in a range between 0 and 540 md / dL were studied. A PBS buffer having pH 7,4 was applied. Area A and absolute sensitivity S of the sensing area 12 can be combined to area sensitivity SA: SA= S / A. Specifically, the following examples were investigated (d – thickness): - SA= 0,2^^(^^ )∙ మ(sensor setup: dHydrogel= about 3µm, dDLM= about 61µm, PEG-DGE amount ^^ ^^in DLM= about wt.3%) - SA= 0,8^^(sensor setup: dHydrogel= around 3µm, dDLM= about 16µm, PEG-DGE amount in DLM= about 11,1wt. %) - SA= 1,5^^(^^ )∙ మ(sensor setup: dHydrogel= around 7,5µm, dDLM= about 13µm, PEG-DGE ^^ ^^amount in DLM= about wt.20%) In Fig.4 to 6, additional experimental results are depicted. Fig.4 shows a graphical representation of normalized area sensitivity SAfor an in vivo analyte sensor in dependence on an amount of PEG-DGE in the membrane layer 25 of the analyte sensor 10. Measuring points 30 for different amount of PEG-DGE in the membrane layer 25 are depicted. A linear fitting curve 31 fits to the measuring points 30. P38942 Fig.5 shows a graphical representation of normalized area sensitivity SAfor an in vivo analyte sensor in dependence on a thickness (d) of the membrane layer 25 of the analyte sensor 10. Measuring points 40 for different layer thickness of the membrane layer 25 are depicted. A linear fitting curve 41 fits to the measuring points 40 indicating: SA~ 1 / d. Layer thickness (d) has been measured by laser scanning microscopy. Fig.6 shows a graphical representation of normalized area sensitivity SAfor an in vivo analyte sensor in dependence on a thickness (d) of a hydrogel-based working electrode of the analyte sensor 10. Measuring points 50 for different thickness of the hydrogel-based working electrode 11 are depicted. A linear fitting curve 51 fits to the measuring points 50. Thickness (d) of hy- drogel-based working electrode (layer thickness) has been measured by laser scanning mi- croscopy.

Claims

P38942 Claims 1. An in vivo analyte sensor (10), comprising: - a substrate (20); - a conductive layer (21) disposed over at least a portion of the substrate (20); - a sensing layer (24) disposed formed on the conductive layer (21), wherein the sens- ing layer (24) forms a sensing area (12) configured to generate one or more signals identifying a monitored analyte level after insertion of the analyte sensor and when maintained in fluid contact with interstitial fluid; and - a membrane layer (25) formed at least on the sensing layer (24); wherein the sensing layer (24) is formed with an area sensitivity of 0,2 to 1,5^^(^^ మ^^ ) ^^within the sensing area (12).

2. Analyte sensor (10) of claim 1, wherein the sensing area (12) is formed with an area size of 0,1 to 1,5 mm2.

3. Analyte sensor (10) of claim 1 or 2, wherein the sensing area (12) is formed with a sen- sitivity of 0,05 to 0,34. Analyte sensor (10) of at least one of the preceding claims, wherein the sensing area (12) is formed as a single spot sensing area (12).

5. Analyte sensor (10) of at least one of the preceding claims, wherein the sensing layer (24) forms a glucose sensing area configured to generate one or more signals identifying a monitored glucose level.

6. Analyte sensor (10) of at least one of the preceding claims, further comprising a working electrode (11), a counter electrode, and a reference electrode, wherein the working elec- trode (11) comprises the sensing area (12).

7. Analyte sensor (10) of claim 6, formed as a two-electrode analyte sensor comprising a combined counter and reference electrode (13),P38942 8. Analyte sensor (10) of claim 6 or 7, further comprising a protecting layer deposited over at least one of the counter electrode, and the reference electrode.

9. Analyte sensor (10) of claim 8, wherein the protecting layer is deposited over a bending portion of the substrate (20).

10. Analyte sensor (10) of at least one of the preceding claims, wherein the membrane layer (25) is made of a flux-limiting polymer membrane material.

11. Analyte sensor (10) of at least one of the preceding claims, wherein the sensing area (12) is located in a sensor tip portion (14).

12. Analyte sensor (10) of at least one of the preceding claims, wherein the substrate (20) is a planar substrate.

13. Analyte sensor (10) of at least one of the preceding claims, wherein the sensing layer (24) is made of an enzymatic Os-hydrogel based sensing material.

14. A method of manufacturing an in vivo analyte sensor (10), comprising: - providing a substrate (20); - disposing a conductive layer (21) over at least a portion of the substrate (20); - disposing a sensing layer (24) on the conductive layer (21) to form a sensing area (12) configured to generate one or more signals identifying a monitored analyte level after insertion of the analyte sensor and when maintained in fluid contact with intersti- tial fluid; and - forming a membrane layer (25) at least on the sensing layer (24); wherein the forming of the sensing layer (24) comprises forming the sensing layer (24) with an area sensitivity of 0,2 to 1,5^^(^^ మwithin the sensing area (12). ^^ ) ^^15. Method of claim 14, wherein at least one of the following layers is formed by a continuous coating process: the conductive layer (21), the sensing layer (24), and the membrane layer (25).

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