Multilayer electrochemical analyte sensors and methods of making and using the same

Abstract

Embodiments of the invention provide multilayer analyte sensors having material layers (eg, high density amine layers) and / or configurations of material layers for enhancing sensor functionality, and methods of making and using such sensors. Typical embodiments of the invention include glucose sensors for diabetes management.

Description

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS

[0002] This application claims priority under Section 120 to U.S. Patent Application No. 15 / 891,264, filed on February 7, 2018, the contents of which are incorporated herein by reference. Technical Field

[0003] The present invention relates to methods and materials that can be used in analyte sensor systems, such as glucose sensors for diabetes management. Background Art

[0004] Sensors are used to monitor a variety of compounds in various environments, including analytes in the body. Quantitative determination of analytes in the human body is of great importance in the diagnosis and maintenance of many pathological conditions. Exemplary analytes that are commonly monitored in a large number of individuals include glucose, lactate, cholesterol, and bilirubin. Determination of glucose concentration in body fluids is particularly important for diabetic individuals who must frequently check the glucose levels in their body fluids to regulate the glucose intake in their diet. The results of such tests are crucial for determining how much (if any) insulin and / or other drugs need to be used.

[0005] Analyte sensors typically include components that convert the interaction with the analyte into a detectable signal, which can be associated with the concentration of the analyte. For example, some glucose sensors use amperometry to monitor glucose in vivo. This amperometric glucose sensor typically includes an electrode coated with glucose oxidase, which is an enzyme that catalyzes the reaction between glucose and oxygen to produce gluconic acid and hydrogen peroxide (H2O2). The H2O2 formed in the reaction changes the electrode current to form a detectable and measurable signal. Based on this signal, the glucose concentration in an individual can be measured.

[0006] A typical electrochemical glucose sensor works based on the following chemical reaction:

[0007]

[0008] H2O2→O2+2H + +2e - Reaction 2

[0009] Glucose oxidase is used to catalyze the reaction between glucose and oxygen to produce gluconic acid and hydrogen peroxide, as shown in reaction 1. H2O2 reacts electrochemically, as shown in reaction 2, and the current is measured by a potentiostat. The stoichiometry of the reaction poses a challenge to the development of in vivo sensors. In particular, to obtain optimal glucose oxidase-based sensor performance, the sensor signal output should be determined solely by the target analyte (glucose) and not by any co-substrate (O2) or kinetic control parameters such as diffusion. If oxygen and glucose are present in equimolar concentrations, then H2O2 is stoichiometrically related to the amount of glucose that reacts with glucose oxidase; and the associated current that produces the sensor signal is proportional to the amount of glucose that reacts with the enzyme. However, if there is not enough oxygen for all the glucose to react with the enzyme, then the current will be proportional to the oxygen concentration, not the glucose concentration. Therefore, in order for a glucose sensor to provide a signal that depends only on the glucose concentration, glucose must be the limiting reagent, that is, the O2 concentration must be in excess for all potential glucose concentrations. However, one problem with using such glucose sensors in vivo is that the oxygen concentration at the site where the sensor is implanted is low relative to glucose, a phenomenon that can compromise the accuracy of the glucose sensor readings (and thus, this phenomenon is referred to as the "oxygen starvation problem").

[0010] Important components of some electrochemical analyte sensors include a material layer disposed above the electrode to enable the sensor to properly measure the analyte signal taking into account oxygen deficiency issues such as those described above. Based on various factors, such as the material components used in the multilayer electrochemical sensor stack and the locations where these components are placed in the stack architecture, the sensors may vary in their stability, reliability, and sensitivity in detecting analyte signals. In this context, there is a need in the art for a multilayer electrochemical sensor with layered materials that is optimized for the production and function of the sensor. There is also a need for a multilayer electrochemical sensor with improved stability, reliability, and sensitivity when detecting analyte signals. The embodiments of the invention disclosed herein meet this need and other needs. Summary of the invention

[0011] Embodiments of the present invention disclosed herein provide electrochemical sensor designs including multilayer analyte sensor stacks. In these embodiments, the components of the multilayer analyte sensor stack are formed by selected layers / materials and are arranged in a stacking architecture in a specific direction, and the specific direction is designed to provide enhanced functional characteristics to these sensors. The present disclosure also provides methods for making and using such sensors. As discussed in detail below, typical embodiments of the present invention relate to the use of sensors that measure the concentration of target aqueous analytes or indicate the concentration of analyte concentrations or substances present in the body (e.g., glucose sensors for diabetes management). Embodiments of the present invention provide innovative ways to simplify the design of certain conventional electrochemical sensors, which have multiple layers arranged above electrodes.

[0012] Illustrative embodiments of the present invention are electrochemical analyte sensors that include a substrate, a working electrode disposed on the substrate, and a multilayer analyte sensor stack disposed on the working electrode. In this embodiment, the multilayer analyte sensor stack includes an analyte sensing layer (e.g., an analyte sensing layer comprising glucose oxidase) disposed directly on the working electrode, and the function of the layer is to detectably change the current at the working electrode in the presence of an analyte. In these embodiments, a high density amine ("HDA") layer comprising a polymer having multiple repeating amine groups (e.g., a poly-L-lysine polymer) is disposed directly on top of the analyte sensing layer. In these embodiments, an analyte modulating layer (e.g., an analyte modulating layer comprising a glucose limiting membrane that modulates the diffusion of glucose from interstitial fluid to the working electrode) is disposed directly on top of the high density amine layer.

[0013] In a typical analyte sensor embodiment, the multilayer analyte sensor stack does not include at least one of: another layer comprising albumin; another layer comprising a silicone adhesion promoter; or a layer comprising glutaraldehyde. For example, in a working embodiment disclosed herein, the multilayer analyte sensor stack consists essentially of an analyte sensing layer, a high-density amine layer, and an analyte modulating layer. In a typical embodiment of the present invention, the high-density amine layer includes a first face in direct contact with the analyte sensing layer and a second face in direct contact with the analyte modulating layer, and the high-density amine layer is used as an adhesive layer to bind the analyte sensing layer to the analyte modulating layer. Optionally, the analyte sensing layer includes a glucose oxidase disposed in the layer so that the analyte sensor senses glucose; and the high-density amine layer also has the function of reducing changes in sensor signals caused by fluctuations in oxygen (O2) levels. In an illustrative working embodiment of the present invention disclosed herein, a polymer having multiple repeating amine groups used to form the high-density amine layer includes a poly-L-lysine polymer having a molecular weight of 30KDa to 300KDa, for example, a molecular weight between 150KDa and 300KDa. Typically, the amount of polymer having multiple repeating amine groups in the high-density amine layer is 0.1 wt% to 0.5 wt%. Optionally, the thickness of the high-density amine layer is 0.1 to 0.4 microns.

[0014] Another embodiment of the present invention is a method of making an electrochemical analyte sensor, comprising the steps of: disposing a working electrode on a substrate; disposing an analyte sensing layer (e.g., an analyte sensing layer comprising glucose oxidase) on the working electrode. These methods further include disposing a high-density amine layer directly on the analyte sensing layer (e.g., using a spray or spin coating process), the high-density amine layer comprising a polymer having multiple repeating amine groups (e.g., a poly-L-lysine polymer); and disposing an analyte modulating layer (e.g., a glucose limiting membrane) directly on the high-density amine layer. Optionally, the electrochemical sensor includes a multilayer analyte sensor stack disposed above the working electrode, the multilayer analyte sensor stack consisting essentially of an analyte sensor layer, a high-density amine layer, and an analyte modulating layer. In the working embodiments disclosed herein, the analyte sensing layer includes glucose oxidase disposed in the layer so that the analyte sensor senses glucose; and the high-density amine layer functions to reduce sensor signal changes caused by fluctuations in oxygen (O2) levels during in vivo glucose sensing.

[0015] Yet another embodiment of the present invention is a method of sensing glucose concentration in a fluid (e.g., interstitial fluid of a diabetic patient or another location where oxygen concentration is low relative to glucose), the method comprising disposing an electrochemical glucose sensor in the fluid, wherein the electrochemical glucose sensor comprises a substrate; a working electrode disposed on the substrate; and a multilayer analyte sensor stack disposed on the working electrode. In these embodiments, the multilayer analyte sensor stack comprises an analyte sensing layer comprising glucose oxidase disposed above the working electrode, which detectably changes the current at the working electrode in the presence of glucose; a high-density amine layer comprising a polymer having multiple repeating amine groups (e.g., a poly-L-lysine polymer), wherein the high-density amine layer is disposed above the analyte sensing layer; and an analyte modulating layer disposed above the high-density amine layer, the analyte modulating layer comprising a material that modulates the diffusion of glucose therethrough. These methods also include monitoring fluctuations in conductivity that occur in the presence of glucose; and correlating the fluctuations in conductivity with the glucose concentration in order to sense the glucose concentration in the fluid. In such embodiments, the high density amine layer functions to increase adhesion between the layers of the multi-layer analyte sensor stack while simultaneously reducing sensor signal variations due to fluctuations in oxygen (O 2 ) levels when sensing glucose concentration in a fluid.

[0016] Those skilled in the art will readily appreciate other objects, features and advantages of the present invention from the following detailed description. However, it should be understood that, although some embodiments of the present invention are indicated, the detailed description and specific embodiments are provided by way of illustration and not limitation. Without departing from the spirit of the present invention, many changes and modifications within the scope of the present invention may be made, and the present invention includes all such modifications.

[0017] BRIEF DESCRIPTION OF THE DRAWINGS

[0018] Figure 1 A schematic diagram illustrating the general structure of a high density amine (HDA) polymer unit that can be used to make a high density amine polymer is provided. R1 includes an alkyl functional group, such as those containing 1-20 carbon atoms. R2 includes a ketone functional group, such as those containing at least one oxygen atom and 1-20 carbon atoms. R3 includes a nitrogen functional group, such as those containing at least one nitrogen atom and 1-20 carbon atoms.

[0019] Figure 2A A schematic diagram is provided showing a conventional (prior art) sensor design comprising an amperometric analyte sensor formed from a plurality of planar layered elements including an albumin layer and an adhesion promoter layer. Figure 2BSchematic diagrams showing the differences between such conventional multilayer sensor stacks and the novel sensor stacks disclosed herein (ie, sensor stacks that do not include a glutaraldehyde-containing layer, a serum albumin-containing layer, or a silicone adhesion promoter-containing layer) are provided.

[0020] Figure 3 Perspective views are provided illustrating a subcutaneous sensor insertion kit, a telemetric characteristic monitor transmitter device, and a data receiving device embodying features of the present invention.

[0021] Figure 4 Schematic diagram of a potentiostat that can be used to measure current in an embodiment of the present invention is shown. Figure 4 As shown, the potentiostat 300 may include an operational amplifier 310 connected in a circuit to have two inputs: Vset and Vmeasure. As shown, Vmeasure is a measurement of the voltage between the reference electrode and the working electrode. On the other hand, Vset is the optimal desired voltage between the working electrode and the reference electrode. The current between the counter electrode and the reference electrode is measured, thereby creating a current measurement result (isig) output from the potentiostat.

[0022] Figure 5 In vivo study data for monitoring glucose in pigs using an amperometric glucose sensor including a layer of HDA material that had been sterilized by ethylene oxide (ETO) is presented. The data demonstrate that after ETO sterilization, the glucose sensor including the HDA material layer exhibits excellent in vivo glucose sensing capabilities for at least 11 days of wear.

[0023] Figure 6 is a graph of data from sensor embodiments having HDA multilayer stacks formed with poly-L-lysine of varying molecular weights. The data demonstrate that sensor embodiments having HDA multilayer stacks have lower baseline oxygen responses than sensor embodiments formed using conventional multilayer stacks.

[0024] Figure 7 Data from an in vivo comparative study of glucose monitoring in pigs using the amperometric glucose sensors disclosed herein that include a layer of HDA material and compared to conventional sensors that do not have an HDA layer (e.g., Figure 2A ) The data from this study show the effectiveness of glucose sensors with HDA polymer layers, which act as adhesion promoters, etc., in electrochemical analyte sensors with multilayer sensor stacks.

[0025] Figure 8Data from an in vitro comparative study that monitored glucose sensing at different O2 concentrations to compare oxygen response in a conventional / conventional glucose oxidase-based sensor and an amperometric glucose sensor disclosed herein comprising a layer of HDA material are shown. These studies demonstrate that the glucose sensor disclosed herein comprising a layer of HDA material exhibits lower in vitro signal changes to changes in oxygen concentration than conventional / conventional glucose oxidase-based sensors. DETAILED DESCRIPTION

[0026] Unless otherwise defined, all technical terms, symbols and other scientific terms or technical terms used herein are intended to have the meanings commonly understood by those skilled in the art to which the present invention belongs. In some cases, for the sake of clarity and / or convenient reference, terms with commonly understood meanings may be defined herein, and including such definitions herein should not necessarily be interpreted as representing substantial differences from those generally understood in the art. Many techniques and procedures described or referenced herein are well understood and commonly used by those skilled in the art using conventional methods. As required, unless otherwise noted, procedures involving the use of commercially available kits and reagents are generally performed according to the schemes and / or parameters defined by the manufacturer. Many terms are defined below.

[0027] All numbers described in the specification and related claims refer to values ​​that can be numerically characterized by values ​​other than integers (e.g., the diameter of a disk), and should be understood to be modified by the term "about". In the case of providing a range of values, it should be understood that each intermediate value between the upper and lower limits of the range, unless otherwise explicitly stated, to one tenth of the unit of the lower limit, and any other specified values ​​or intermediate values ​​in the range are included in the present invention. The upper and lower limits of these smaller ranges can be independently included in the smaller ranges, and are also included in the present invention, subject to any specific exclusive restrictions within the range. When the stated range includes one or two limits, the range excluding any one or both of those included limits is also included in the present invention. In addition, all publications mentioned herein are incorporated herein by reference, thereby disclosing and describing methods and / or materials related to the cited publications. The publications cited herein are cited for their disclosures before the filing date of this application. Nothing herein shall be interpreted as admitting that the inventor has no right to advance the date of the publication by virtue of an earlier invention priority date or a prior invention date. In addition, the actual publication date may be different from the date shown and needs to be verified separately.

[0028] As used herein, the term "analyte" is a broad term and is used in its general sense, including but not limited to referring to a substance or chemical composition in a fluid such as a biological fluid (e.g., blood, interstitial fluid, cerebrospinal fluid, lymph or urine) that can be analyzed. The analyte may include naturally occurring substances, artificial substances, metabolites and / or reaction products. In a common embodiment, the analyte is glucose. However, embodiments of the present invention can be used together with sensors designed to detect a variety of other analytes. Illustrative analytes include but are not limited to lactate and naturally occurring salts, sugars, proteins, fats, vitamins and hormones in the body (e.g., in blood or interstitial fluid). The analyte may be naturally present in a biological fluid or endogenous, for example, metabolites, hormones, antigens, antibodies, etc. Alternatively, the analyte may be introduced into the human body or exogenous, for example, contrast agents, radioisotopes, chemical reagents, synthetic blood or drugs or pharmaceutical compositions based on fluorocarbons for imaging, including but not limited to insulin. Metabolites of drugs and pharmaceutical compositions are also covered analytes.

[0029] The term "sensor" is used in its general sense, such as in "analyte sensor", and includes, but is not limited to, devices for detecting compounds such as analytes. A "sensor system" includes, for example, elements, structures, and architectures designed to facilitate sensor use and function (e.g., specific 3-dimensional constellations of elements). A sensor system may include, for example, a composition such as a material layer having selected properties, such as a high-density amine layer formed from a polymer having multiple repeating amine groups (e.g., a high-density amine layer containing 0.1 wt % to 0.5 wt % poly-L-lysine having a molecular weight between 150 KDa and 300 KDa), and electronic components, such as elements and devices for signal detection and analysis (e.g., current detectors, monitors, processors, etc.).

[0030] As used herein, the terms "electrochemical reaction surface" and "electroactive surface" are broad terms and are used in their general sense, including but not limited to the surface of an electrode where an electrochemical reaction occurs. In one example, the working electrode measures hydrogen peroxide produced by an enzyme-catalyzed reaction of the analyte being detected, thereby generating an electric current (e.g., the detection of glucose analyte utilizes glucose oxidase to produce a byproduct H2O2, which reacts with the surface of the working electrode to produce two protons (2H + ), two electrons (2e - ) and an oxygen molecule (O2), which produces the electronic current that is detected). In the case of the counter electrode, the reducible species such as O2 is reduced at the electrode surface to balance the current produced by the working electrode.

[0031] As discussed in detail below, embodiments of the present invention relate to the use of electrochemical sensors that measure the concentration of a target analyte or indicate the concentration of an analyte or a substance present in a fluid. In some embodiments, the sensor is a continuous device, such as a subcutaneous, transdermal, or intravascular device. In some embodiments, the device can analyze multiple intermittent blood samples. The sensor embodiments disclosed herein can use any known method, including invasive, minimally invasive, and non-invasive sensing techniques, to provide an output signal indicating the concentration of a target analyte. The product is then measured using an electrochemical method, and the output of the electrode system is therefore used as a measure of the analyte.

[0032] Embodiments of the invention disclosed herein provide sensors of the type used, for example, in subcutaneous or transcutaneous monitoring of blood glucose levels in diabetic patients. A variety of implantable electrochemical biosensors have been developed to treat diabetes and other life-threatening diseases. Many existing sensor designs use some form of immobilized enzyme to achieve their biospecificity. Embodiments of the invention described herein may be applicable and implemented in a variety of known electrochemical sensors, including, for example, U.S. Patent Application No. 20050115832, U.S. Patent Nos. 6,001,067, 6,702,857, 6,212,416, 6,119,028, 6,400,974, 6,595,919, 6,141,573, 6,122,536, 6,512,939, 5,605,152, 4,431,004, 4,703,756, 6,514,718, 5,985,129, 5,390,691, 5,391,250, 5,482,473, 5,299,571, 5,568,806, 5,494,562, 6,120,676, 6,542,765, 7,033,336 and PCT International Publication No. WO 01 / 58348, WO04 / 021877, WO 03 / 034902, WO 03 / 035117, WO 03 / 035891, WO 03 / 023388, WO 03 / 022128, WO 03 / 022352, WO 03 / 023708, WO 03 / 036255, WO 03 / 036310, WO 08 / 042,625 and WO 03 / 074107, and European Patent Application No. EP 1153571, the contents of each of which are incorporated herein by reference.

[0033] Illustrative embodiments and related features of the present invention

[0034] Embodiments of the present invention disclosed herein provide sensors designed to include a multilayer analyte sensor stack formed of selected materials, which provides enhanced functionality and / or material properties for the sensor. The present disclosure also provides methods for making and using such sensors. As discussed in detail below, typical embodiments of the present invention relate to the use of sensors that measure the concentration of a target aqueous analyte or indicate the concentration of an analyte in vivo or the concentration of a substance present. In some embodiments, the sensor is a subcutaneous, intramuscular, intraperitoneal, intravascular or transcutaneous device. Typically, the sensor can be used for continuous analyte monitoring. Embodiments of the sensors disclosed herein can use any known method, including invasive, minimally invasive and non-invasive sensing techniques, to provide an output signal indicating the concentration of a target analyte. Embodiments of the present invention provide an innovative way to simplify the design of conventional electrochemical sensors with multiple layers disposed above a working electrode.

[0035] Compared with conventional multilayer electrochemical sensor designs (e.g. Figure 2A Compared to the methods disclosed in , embodiments of the present invention having clusters of components including high density amine layers exhibit many advantages. For example, embodiments of the present invention have fewer layers of material, a feature that can be used to simplify the manufacturing process and reduce variability between sensors compared to conventional processes for making analyte sensor stacks that utilize multi-component adhesive layers to "glue" the GLM to the GOx (where the layers are generated in situ by many chemical reactions occurring simultaneously, resulting in variability between sensors). Figure 2B , used to compare conventional analyte sensors and the HDA sensors disclosed herein. Another advantage is that certain HDA sensor embodiments disclosed herein do not contain human serum albumin (HSA). Other advantages of embodiments of the present invention include improved stability with the elimination of glutaraldehyde (glutaraldehyde cross-linked glucose oxidase in conventional sensor designs can reduce GOx activity and stability). By eliminating glutaraldehyde, we eliminate this potential cause of sensor instability. Another related advantage is that sensor embodiments disclosed herein were observed to exhibit more robust sterilization properties in electron beam and ETO processes.

[0036] Other related advantages of the unique cluster of layered materials disposed on the working electrode(s) disclosed herein include more robust layer surfaces and improved stability relative to arrangements of different initialization profiles (sensors including HDA layers are stable over a range of different initialization profiles). Other advantages may include, for example, more uniform layers and more sites for electrochemical reactions, features that contribute to the stability and / or sensitivity of the layered sensor structure. For example, by reducing the likelihood that one or more layers of material may delaminate, a smoother and more adherent surface is provided, thereby contributing to sensor stability. Importantly, a key advantage is the improved oxygen response observed in glucose oxidase-based sensors formed with the HDA layer, with the HDA poly-L-lysine sensor showing less signal variation under variable oxygen concentrations (5% to 1%). This feature addresses the oxygen starvation problem of glucose sensors discussed above (e.g., Figure 6 and 8 ). Then, Figure 7 Data from an in vivo comparative study using an amperometric glucose sensor including a layer of HDA material as disclosed herein to monitor glucose in pigs is shown, compared to conventional sensors (e.g., Figure 2A ) for comparison.

[0037] In the high density amine layer disclosed herein, the polymer having multiple repeating amine groups can adopt a variety of configurations. The simplest polymer architecture having multiple repeating amine groups is a linear chain: a single main chain without branches. Alternatively, the polymer can be branched. Branched polymer molecules consist of a main chain with one or more substituent side chains or branches. Special types of branched polymers include dendrimers. Dendrimers are a special case of macromolecules in which each monomer unit is branched. In some embodiments of the present invention, the polymer having multiple repeating amine groups within the HDA layer exhibits a linear and / or branched and / or dendrimer-like structure. In illustrative embodiments of the present invention disclosed herein, the HDA layer comprises a poly-L-lysine polymer.

[0038] Although the illustrative working embodiments of the present invention are formed from linear polymers, polymers having multiple repeating amine groups within the HDA layer may exhibit linear, branched, and / or dendrimer-like structures. Such HDA polymers include, for example, poly-L-lysine, poly-D-lysine, chitosan, aminodextran, polyethyleneimine, other poly-L-amino acid polymers, and the like. In certain embodiments of the present invention, the polymer includes the general structure shown below having R1, R2, and R3, wherein:

[0039]

[0040] R 1 =Alkyl functional groups of various chain lengths (straight and / or branched)

[0041] R 2 = Ketone functional group

[0042] R 3 = Nitrogen functional group

[0043] In certain embodiments, the polymer comprises poly-L-lysine units:

[0044]

[0045] In a specific illustrative embodiment, the polymer comprises a molecular structure such as:

[0046]

[0047] In another specific illustrative embodiment, the polymer comprises a molecular structure such as:

[0048]

[0049] The high density amine layer can be formed according to methods recognized in the art, for example, by weighing out an appropriate amount of a polymer such as poly-L-lysine, dissolving the amount of poly-L-lysine in an appropriate amount of water to form a clear solution and stirring for 1 hour. The technician can then use this solution to prepare a 0.1 to 0.5 wt%

[0050] (w / w %) of a high density amine composition to form a layer in the sensor disclosed herein. Typically, the layer is applied to the sensor stack by spraying the poly-L-lysine solution onto the substrate several times (e.g., 3 times, with repeated spraying cycles of the biological points), such that more repeated applications = more material is deposited onto the substrate. In the working embodiments disclosed herein, poly-L-lysine polymers with different molecular weights were examined, where HMW = high molecular weight = 150 to 300 KDa, MMW = medium molecular weight = 70 to 150 KDa, and LMW = low molecular weight = 30 to 70 KDa.

[0051] An illustrative embodiment of the invention is an electrochemical analyte sensor comprising a substrate, a working electrode disposed on the substrate, and a multilayer analyte sensor stack disposed on the working electrode. In this embodiment, the multilayer analyte sensor stack comprises an analyte sensing layer disposed directly on the working electrode, wherein the analyte sensing layer detectably changes the current at the working electrode in the presence of an analyte; a high-density amine layer disposed on the analyte sensing layer, wherein the high-density amine layer comprises a poly-L-lysine polymer; and an analyte modulating layer (e.g., a glucose limiting membrane) disposed on the high-density amine layer, wherein the analyte modulating layer modulates diffusion of an analyte (e.g., glucose) from an external environment (e.g., interstitial fluid) to the working electrode.

[0052] In such an analyte sensor embodiment, the multilayer analyte sensor stack does not include at least one of: another layer comprising albumin (and optionally a sensor layer without albumin); another layer comprising a silicone adhesion promoter; or another layer comprising glutaraldehyde (and optionally a sensor layer without glutaraldehyde or a sensor layer without a glutaraldehyde portion). For example, in a working embodiment disclosed herein, the multilayer analyte sensor stack consists essentially of an analyte sensing layer, a high-density amine layer, and an analyte modulating layer. In a typical embodiment of the present invention, the high-density amine layer includes a first side that is in direct contact with the analyte sensing layer and a second side that is in direct contact with the analyte modulating layer, the contact causing the layer to act as an adhesive layer, binding the analyte sensing layer to the analyte modulating layer. Optionally, the analyte sensing layer includes glucose oxidase disposed in the layer so that the analyte sensor senses glucose; and the high-density amine layer also has the function of reducing changes in sensor signals caused by fluctuations in O2 levels (see, e.g. Figure 6 Data from illustrative embodiments of the invention are shown). Polymers having multiple repeating amine groups within the HDA layer exhibit linear and / or branched and / or dendrimer-like structures. In certain embodiments of the invention, the poly-L-lysine in the high-density amine layer has a molecular weight between 30 KDa and 300 KDa, for example, a molecular weight between 150 KDa and 300 KDa. Typically, the amount of poly-L-lysine in the high-density amine layer is 0.1 wt % to 0.5 wt %. Optionally, the thickness of the high-density amine layer is 0.1 to 0.4 microns. These 0.1 to 0.4 micron thin adhesive layers have unexpected advantages because they exhibit lower oxygen responses and faster hydration times than conventional sensors that do not have such thin HDA layers.

[0053] Another embodiment of the invention is a method of making an electrochemical analyte sensor comprising the steps of: disposing a working electrode on a substrate; disposing an analyte sensing layer on the working electrode, wherein the analyte sensing layer detectably changes the current at the working electrode in the presence of an analyte; disposing a high density amine layer comprising, for example, an HDA polymer directly on the analyte sensing layer (e.g., using a spray coating process); and disposing an analyte modulating layer directly on the high density amine layer, wherein the analyte modulating layer modulates the diffusion of the analyte therethrough, thereby making an electrochemical analyte sensor. Optionally, the electrochemical sensor comprises a multilayer analyte sensor stack disposed above the working electrode, the multilayer analyte sensor stack consisting essentially of the analyte sensor layer, the high density amine layer, and the analyte modulating layer. An illustrative poly-L-lysine solution used to make an embodiment of the invention is 150 to 300 KDa poly-L-lysine, 0.3 poly-L-lysine w / w%, which is applied in about 2 spray repetitions. In a working embodiment disclosed herein, the analyte sensing layer includes glucose oxidase disposed therein so that the analyte sensor senses glucose; and the high-density amine layer also has the function of reducing sensor signal changes caused by fluctuations in oxygen (O2) levels during glucose sensing.

[0054] One unexpected advantage of analyte sensors including high-density amine layers disclosed herein is that they are able to maintain excellent functionality (i.e., analyte sensing) after sterilization by ethylene oxide. Specifically, when devices such as analyte sensors are sterilized with ethylene oxide, problems arise if the ethylene oxide reacts with one or more sensitive components of the device (such as glucose oxidase in an amperometric glucose sensor) and inhibits its activity. Such problems may prevent the effective use of ethylene oxide sterilization procedures on such devices. Therefore, it is desirable to design methods and materials (e.g., high-density amine layers disclosed herein) to address such challenges in the art. In this case, embodiments of the present invention include methods for making analyte sensors including high-density amine layers disclosed herein, the methods including the step of sterilizing the sensor with ethylene oxide; sensors including high-density amine layers disclosed herein that have been sterilized with ethylene oxide; and methods of sensing analytes such as glucose with these sensors including high-density amine layers disclosed herein, which have been sterilized with ethylene oxide. Various ethylene oxide sterilization procedures are known in the art (see, for example, US Patent Publications 20120252125, 20110233068, 20070292305, and 20050089442, the contents of which are incorporated herein by reference). Illustrative parameters for ethylene oxide are as follows.

[0055] parameter level unit Ethylene oxide concentration 200-800 mg / L humidity >＝30 %RH temperature <＝140 F Dwell time 2-12 Hour CO2 mixture <＝80％ %composition

[0056] Figure 5In vivo research data for monitoring glucose in pigs using an amperometric glucose sensor including a layer of HDA material that has been sterilized by ETO according to such ETO sterilization parameters are presented. The data show that after ETO sterilization, the glucose sensor including a layer of HDA material exhibits excellent in vivo glucose sensing capabilities for at least 11 days of wear.

[0057] Yet another embodiment of the present invention is a method of sensing glucose concentration in a fluid (e.g., an environment in which the glucose concentration is low relative to the oxygen concentration), comprising disposing an electrochemical glucose sensor in the fluid, wherein the electrochemical glucose sensor comprises a substrate; a working electrode disposed on the substrate; and a multilayer analyte sensor stack disposed on the working electrode. In these embodiments, the multilayer analyte sensor stack comprises an analyte sensing layer comprising glucose oxidase disposed above the working electrode, wherein the analyte sensing layer detectably changes the current at the working electrode in the presence of an analyte. The stack also comprises a high-density amine layer, such as a layer comprising a poly-L-lysine polymer, wherein the high-density amine layer is disposed above the analyte sensing layer; and an analyte regulating layer disposed on the high-density amine layer, wherein the analyte regulating layer regulates the diffusion of glucose therethrough. These methods also include monitoring fluctuations in conductivity that can be observed when glucose reacts with glucose oxidase; and correlating fluctuations in conductivity with glucose concentration to sense glucose concentration in the fluid. In such an embodiment, the high density amine layer acts to increase the adhesion between the layers of the multilayer analyte sensor stack while reducing sensor signal changes caused by fluctuations in oxygen (O2) levels when sensing glucose concentration in a fluid. Optionally, the analyte modulating layer is a glucose limiting membrane comprising a polyurethane / polyurea polymer formed from a mixture comprising a diisocyanate, a hydrophilic polymer containing a hydrophilic diol or a hydrophilic diamine, and a siloxane having amino, hydroxyl or carboxylic acid functional groups at the end.

[0058] In typical embodiments of the invention, the electrochemical sensor is operably coupled to a sensor input capable of receiving a signal from the electrochemical sensor; and a processor coupled to the sensor input, wherein the processor is capable of characterizing one or more signals received from the electrochemical sensor. In certain embodiments of the invention, the electrical conduit of the electrode is coupled to a potentiostat (see, e.g., Figure 4). Optionally, a pulse voltage is used to obtain a signal from the electrode. In a typical embodiment of the invention, the processor is capable of comparing a first signal received from the working electrode in response to a first working potential with a second signal received from the working electrode in response to a second working potential. Optionally, the electrode is coupled to a processor adapted to convert data obtained by observing fluctuations in the current from a first format to a second format. Such embodiments include, for example, a processor designed to convert a sensor current input signal (e.g., ISIG measured in nA) to a blood glucose concentration.

[0059] In many embodiments of the present invention, the sensor includes a biocompatible region suitable for implantation in vivo. In some embodiments, the sensor includes a discrete probe that pierces the in vivo environment. In an embodiment of the present invention, the biocompatible region may include a polymer that contacts tissue in vivo. Optionally, the polymer is a hydrophilic polymer (e.g., a polymer that absorbs water). In this way, the sensor used in the system of the present invention can be used to sense a variety of analytes in different aqueous environments. In some embodiments of the present invention, an electrode is coupled to a puncture member (e.g., a needle) suitable for implantation in vivo. Although the sensor embodiment of the present invention may include one or two puncture members, optionally, this type of sensor device may include 3 or 4 or 5 or more puncture members, which are coupled to a substrate element and extend therefrom, and are operably coupled to 3 or 4 or 5 or more electrochemical sensors (e.g., microneedle arrays, embodiments of which are disclosed in, for example, U.S. Patents 7,291,497 and 7,027,478, and U.S. Patent Application No. 20080015494, the contents of which are incorporated herein by reference).

[0060] In some embodiments of the invention, the device includes one or more working electrodes, counter electrodes, and reference electrodes, optionally, they are aggregated together in a unit consisting essentially of one working electrode, one counter electrode, and one reference electrode; and the aggregated unit is longitudinally distributed on the base layer in a repeating unit pattern. In some sensor embodiments, the distributed electrodes are organized / set within a flexible circuit assembly (i.e., a circuit assembly that utilizes flexible rather than rigid materials). Such flexible circuit assembly embodiments provide an interconnect assembly of elements (e.g., electrodes, electrical conduits, contact pads, etc.) that is configured to contribute to the comfort of the wearer (e.g., by reducing pad stiffness and wearer discomfort).

[0061] As described above, the sensor electrodes of the present invention are coated with a variety of materials having properties that, for example, facilitate analyte sensing. In a typical embodiment of the present invention, the analyte sensing layer is directly disposed on the working electrode and includes a reagent selected for its ability to detectably change the current at the working electrode in the presence of an analyte. In the working embodiments of the present invention disclosed herein, the reagent is glucose oxidase, a protein that undergoes a chemical reaction in the presence of glucose, the chemical reaction resulting in a change in the current at the working electrode. These working embodiments also include an analyte modulating layer disposed above the analyte sensing layer, wherein the analyte modulating layer modulates the diffusion of glucose when glucose migrates from the in vivo environment to the analyte sensing layer. In certain embodiments of the present invention, the analyte modulating layer comprises a hydrophilic comb copolymer having a central chain and a plurality of side chains coupled to the central chain, wherein at least one side chain comprises a siloxane moiety. In certain embodiments of the present invention, the analyte modulating layer comprises a blended mixture of a linear polyurethane / polyurea polymer and a branched acrylate polymer; and the linear polyurethane / polyurea polymer and the branched acrylate polymer are blended in a ratio of 1:1 to 1:20 (e.g., 1:2) weight %. Typically, the analyte modulating layer composition includes a first polymer formed from a mixture comprising a diisocyanate; at least one hydrophilic diol or hydrophilic diamine; and a siloxane; the first polymer is blended with a second polymer formed from a mixture comprising: 2-(dimethylamino)ethyl methacrylate; methyl methacrylate; monomethacryloxypropyl polydimethylsiloxane; poly(ethylene oxide) methyl ether methacrylate; and 2-hydroxyethyl methacrylate. As disclosed herein, additional material layers may be included in such a device. For example, in a typical embodiment of the invention, the device includes a high-density amine layer disposed between and in direct contact with the analyte sensing layer and the analyte modulating layer to exhibit a number of beneficial properties, including the ability to provide a smoother surface structure and further promote adhesion between the analyte sensing layer and the analyte modulating layer. Without being bound by a particular scientific theory or mechanism of action, it is believed that the adhesion between the layers is promoted by: a smoother layer contact architecture, and van der Waals interactions between the HDA polymers in the HDA layer and compounds present in the analyte sensing layer disposed on a first side of the HDA layer, and van der Waals interactions between the HDA polymers and compounds present in the analyte modulating layer disposed on a second side of the HDA layer (i.e., giving the HDA layer a "sandwich" configuration).

[0062] Figure 2AOne prior art conventional sensor embodiment shown in is an amperometric sensor 100 having multiple layered elements, which includes a base layer 102, a conductive layer 104 (e.g., a conductive layer including multiple conductive members) disposed on and / or coupled to the base layer 102. The following comments relate to this conventional sensor, which are described to help understand the differences between such conventional sensors and the present invention disclosed herein. Typically, the conductive layer 104 includes one or more electrodes. The analyte sensing layer 110 (typically including an enzyme, such as glucose oxidase) is disposed on one or more exposed electrodes of the conductive layer 104. A protein layer 116 is disposed on the analyte sensing layer 110. An analyte regulating layer 112 is disposed above the analyte sensing layer 110 to regulate the proximity of an analyte (e.g., glucose) to the analyte sensing layer 110. As Figure 2A As shown, an adhesion promoter layer 114 is disposed between layers such as the analyte modulating layer 112 and the analyte sensing layer 110 to facilitate their contact and / or adhesion. This embodiment also includes a covering layer 106, such as a polymer coating that can be disposed on a portion of the sensor 100. The hole 108 can be formed in one or more layers of this type of sensor. Amperometric glucose sensors with this type of design are disclosed, for example, in U.S. Patent Application Publication Nos. 20070227907, 20100025238, 20110319734, and 20110152654, the contents of each of which are incorporated herein by reference. Figure 2B A comparison between these conventional multi-layer sensor stacks and the present invention disclosed herein (ie, a multi-layer sensor stack including an HDA layer 500) is shown.

[0063] As described above, embodiments of the present invention also include methods for making and using the HDA multilayer sensor stacks disclosed herein. Yet another embodiment of the present invention is a method for sensing an analyte in a mammal. Typically, the method includes implanting an analyte sensor having an HDA multilayer sensor stack into a mammal (e.g., in the interstitial space of a diabetic individual), sensing a change in current at a working electrode in the presence of an analyte; and then correlating the change in current with the presence of an analyte, such that the analyte is sensed.

[0064] Embodiments of the present invention also provide articles and kits for observing analyte concentrations. In illustrative embodiments, the kit includes a sensor, the sensor including a HDA multilayer sensor stack as described herein. In a typical embodiment, the sensor is disposed in a kit in a sealed sterile dry package. Optionally, the kit includes an insertion device that facilitates sensor insertion. The kit and / or sensor kit typically include a container, a label, and an analyte sensor as described above. Suitable containers include, for example, easy-to-open packaging made of materials such as metal foil, bottles, vials, syringes, and test tubes. The container can be formed of a variety of materials, such as metal (e.g., foil) paper products, glass, or plastic. A label on or associated with the container indicates that the sensor is used to analyze a selected analyte. The kit and / or sensor kit may include other materials desired from a commercial and user perspective, including buffers, diluents, filters, needles, syringes, and packaging inserts with instructions for use.

[0065] Specific aspects of embodiments of the invention are discussed in detail in the following sections.

[0066] Typical components, configurations, and analyte sensor embodiments of the present invention

[0067] A. Typical Elements Present in Embodiments of the Invention

[0068] The invention disclosed herein includes compositions containing high density amine (HDA) polymers that can be used in multilayer electrochemical sensor stacks as a means of imparting functional advantages to the sensor. Figure 1 The schematic diagram provided illustrates the general structure of such polymers that can be used to prepare these polymers.

[0069] Figure 2A A cross section of a conventional sensor embodiment 100 is shown. Components of the sensor are typically characterized herein as layers of the multilayer electrochemical sensor stack because, for example, it can be easily characterized as Figure 2A The conventional sensor structure shown in Figure 2B The distinction of the present invention disclosed herein as shown in FIG. 5 is a diagram of a sensor (i.e., a sensor including an HDA layer 500). A skilled person will appreciate that in certain embodiments of the present invention, sensor components are combined such that multiple components form one or more heterogeneous layers. In this case, a skilled person will appreciate that although certain layers / components of conventional sensor embodiments may be used in the HDA sensors disclosed herein, the arrangement and composition of the layered components are very different in the HDA sensor embodiments of the present invention. A skilled person will appreciate that in certain embodiments, the present invention includes elements / layers present in conventional sensors, excludes other elements, and / or includes new material layers / elements. For example, Figure 2ACertain elements disclosed in the invention may also be present in the invention disclosed herein (e.g., a base layer, an analyte sensing layer, an analyte modulating layer, etc.), and Figure 2B As shown, other elements are not present (e.g., a separate HSA protein layer, a layer comprising a silicone adhesion promoter, etc.). Similarly, embodiments of the present invention include layers / elements having materials arranged in a unique configuration that are not present in conventional sensors (e.g., a high density amine (HDA) polymer layer 500).

[0070] Figure 2A The conventional embodiment shown includes a substrate 102 that supports the sensor 100. The substrate 102 can be made of a material such as a metal and / or a ceramic and / or a polymer substrate, which can be self-supporting or further supported by another material known in the art. Embodiments may include a conductive layer 104 disposed on and / or combined with the substrate 102. Typically, the conductive layer 104 includes one or more conductive elements that function as electrodes. The operating sensor 100 typically includes a plurality of electrodes, such as a working electrode, a counter electrode, and a reference electrode. Other embodiments may also include a plurality of working electrodes and / or counter electrodes and / or reference electrodes and / or one or more electrodes that perform multiple functions, such as an electrode that functions as both a reference electrode and a counter electrode.

[0071] As discussed in detail below, many known techniques and materials can be used to produce the base layer 102 and / or the conductive layer 104. In certain embodiments of the present invention, the circuit of the sensor is defined by etching the conductive layer 104 provided into a desired conductive path pattern. A typical circuit for the sensor 100 includes two or more adjacent conductive paths having an area at the proximal end to form a contact pad and an area at the distal end to form a sensor electrode. An electrically insulating cover layer 106 such as a polymer coating can be provided on a portion of the sensor 100. Acceptable polymer coatings used as an insulating protective cover layer 106 can include, but are not limited to, non-toxic biocompatible polymers such as silicone compounds, polyimides, biocompatible solder masks, epoxy acrylate copolymers, and the like. In the sensor of the present invention, one or more exposed areas or holes 108 can be formed through the cover layer 106 to open the conductive layer 104 to the external environment and, for example, allow an analyte such as glucose to penetrate the layer of the sensor and be sensed by the sensing element. The hole 108 can be formed by a variety of techniques, including laser ablation, masking with a mask, chemical grinding or etching, or photolithographic development, and the like. In certain embodiments of the present invention, during fabrication, a second photoresist may also be applied to the protective layer 106 to define areas of the protective layer to be removed to form one or more holes 108. The exposed electrodes and / or contact pads may also undergo secondary processing (e.g., through the holes 108), such as additional plating processes, to prepare the surface and / or enhance the conductive areas.

[0072] exist Figure 2A In the conventional sensor configuration shown in , the analyte sensing layer 110 is disposed on one or more exposed electrodes of the conductive layer 104. Typically, the analyte sensing layer 110 is an enzyme layer. Most typically, the analyte sensing layer 110 includes an enzyme capable of producing and / or utilizing oxygen and / or hydrogen peroxide, such as glucose oxidase. Optionally, the enzyme in the analyte sensing layer is combined with a second carrier protein such as human serum albumin, bovine serum albumin, etc. In an illustrative embodiment, an oxidoreductase such as glucose oxidase in the analyte sensing layer 110 reacts with glucose to produce hydrogen peroxide, which then regulates the current at the electrode. Since this regulation of the current depends on the concentration of hydrogen peroxide, and the concentration of hydrogen peroxide is related to the concentration of glucose, the concentration of glucose can be determined by monitoring this regulation of the current. In a specific embodiment of the present invention, hydrogen peroxide is oxidized on a working electrode (also referred to herein as an anode working electrode) as an anode, and the resulting current is proportional to the concentration of hydrogen peroxide. This modulation of the current caused by changes in hydrogen peroxide concentration can be monitored by any of a variety of sensor detector devices, such as a Universal Sensor amperometric biosensor detector or one of a variety of other similar devices known in the art, such as the glucose monitoring device produced by Medtronic Diabetes.

[0073] In an embodiment of the invention, the analyte sensing layer 110 can be applied over a portion of the conductive layer or over the entire area of ​​the conductive layer. Typically, the analyte sensing layer 110 is disposed on a working electrode, which can be a positive electrode or a negative electrode. Optionally, the analyte sensing layer 110 is also disposed on a counter electrode and / or a reference electrode. Methods for producing a thin analyte sensing layer 110 include brushing the layer onto a substrate (e.g., a reactive surface of a platinum black electrode), as well as spin coating processes, dip coating and drying processes, low shear spray processes, inkjet printing processes, screen processes, and the like.

[0074] In this case, a variety of spin coating materials and methods are known in the art (see, for example, Sahu et al., Indian J. Phys. 83(4)493-502 (2009), and U.S. Patent Publications 20020127878, 20020127878, 20090285982, and 20140272704). In certain embodiments of the present invention, a material comprising a high density amine layer of a polymer having multiple repeating amine groups (e.g., a poly-L-lysine polymer) is mixed with another material such as a solvent or other agent that adjusts the viscosity of the solution in order to optimize spin coating uniformity. In this case, in order to prepare a HDA layer for spin coating, a viscosity modifier and / or one or two or more solvents can be mixed together. For example, when two solvents are used, a major component of something that evaporates relatively quickly and a minor component of something that evaporates relatively slowly can be used. By using this combination, it is often possible to optimize various aspects of the process because during the spin coating process, the primary component evaporates quickly to provide good coverage and uniformly thick films, while the remaining secondary component still leaves enough plasticity to organize the molecules before the film is completely dry.

[0075] The analyte modulating membrane layer 112 may include a glucose limiting membrane that modulates the amount of glucose that contacts an enzyme such as glucose oxidase present in the analyte sensing layer. Such a glucose limiting membrane may be made of a variety of materials known to be suitable for such purposes, such as silicone compounds such as polydimethylsiloxane, polyurethane, polyurea cellulose acetate, Nafion, polyester sulfonic acid (e.g., Kodak AQ), hydrogels, or any other suitable hydrophilic membrane known to those skilled in the art.

[0076] In a typical embodiment of the present invention, Figure 2BAs shown, a material layer including a high-density amine composition layer 500 is disposed between the analyte modulating layer 112 and the analyte sensing layer 110 to facilitate their contact and / or adhesion. In a typical embodiment of the present invention, the high-density amine layer 500 includes a first side in direct contact with the analyte sensing layer and a second side in direct contact with the analyte modulating layer, and serves as an adhesive layer for bonding the analyte sensing layer to the analyte modulating layer. Optionally, the analyte sensing layer includes a glucose oxidase disposed in the layer so that the analyte sensor senses glucose; and the high-density amine layer 500 also has the function of reducing the sensor signal changes caused by fluctuations in oxygen (O2) levels. In certain embodiments of the present invention, the poly-L-lysine in the high-density amine layer 500 has a molecular weight between 30KDa and 300KDa, for example, a molecular weight between 150KDa and 300KDa. Typically, the amount of poly-L-lysine in the high-density amine layer 500 is 0.1 wt % to 0.5 wt %. Optionally, high density amine layer 500 is 0.1 to 0.4 microns thick.

[0077] Typical Analyte Sensor Components of the Invention

[0078] The following disclosure provides examples of typical elements / ingredients used in sensor embodiments of the present invention. Although these elements can be described as discrete units (e.g., layers), it is understood by those skilled in the art that sensors can be designed to include elements having some or all of the material properties and / or combinations of functions of the elements / ingredients discussed below (e.g., elements are used as both supporting substrate components and / or conductive components and / or matrices for analyte sensing components, and further as electrodes in sensors). It is understood by those skilled in the art that selected elements from these thin film analyte sensors can be suitable for many sensor systems, such as those described below.

[0079] Base ingredients

[0080] The sensors of the present invention typically include a substrate component (see, e.g. Figure 2A 102 in the device). The term "substrate component" is used herein according to the recognized professional terminology in the art and refers to a component in a device that typically provides a supporting matrix for multiple components that are stacked on top of each other and include a functional sensor. In one form, the substrate component includes a thin film sheet of insulating (e.g., electrically insulating and / or impermeable) material. This substrate component can be made of various materials with desired qualities (such as dielectric properties, impermeability and air tightness). Some materials include metals and / or ceramics and / or polymer substrates, etc.

[0081] Conductive components

[0082] The electrochemical sensors of the present invention typically include a conductive component disposed on a substrate component, the conductive component including at least one electrode for contacting an analyte to be analyzed or its byproducts (e.g., oxygen and / or hydrogen peroxide) (see, e.g., Figure 2A 104 in the analyte sensing component 110). The term "conductive component" is used herein according to the recognized terminology in the art. An illustrative example thereof is a conductive component forming a working electrode that can measure an increase or decrease in current in response to exposure to a stimulus (such as a change in concentration of an analyte or its byproducts) compared to a reference electrode that does not experience a change in analyte concentration, the analyte being a co-reactant (such as oxygen) used when the analyte interacts with a composition (such as the enzyme glucose oxidase) present in the analyte sensing component 110, or the analyte is a reaction product of this interaction (such as hydrogen peroxide). Illustrative examples of such elements include electrodes that are capable of generating a variable detectable signal in the presence of a variable concentration of a molecule such as hydrogen peroxide or oxygen.

[0083] In addition to the working electrode, the analyte sensor of the present invention typically also includes a reference electrode or a combined reference and counter electrode (also referred to as a quasi-reference electrode or counter / reference electrode). If the sensor does not have a counter / reference electrode, it may include a separate counter electrode, which may be made of the same or different material as the working electrode. A typical sensor of the present invention has one or more working electrodes and one or more counter, reference and / or counter / reference electrodes. An embodiment of the sensor of the present invention has two, three or four or more working electrodes. These working electrodes in the sensor may be connected as a whole or may be kept separate. Alternatively, the electrode may be arranged on a single surface or one side of the sensor structure. Alternatively, the electrode may be arranged on multiple surfaces or sides of the sensor structure (and, for example, may be connected to the surface on which the electrode is arranged by passing through a through hole of the sensor material). In certain embodiments of the present invention, the reactive surface of the electrode has different relative areas / sizes, such as a 1X reference electrode, a 2.6X working electrode, and a 3.6X counter electrode.

[0084] Analyte sensing components

[0085] The electrochemical sensor of the present invention includes an analyte sensing component disposed on an electrode of the sensor (see, e.g. Figure 2A110 in). The term "analyte sensing component" is used herein according to professional terms recognized in the art, and refers to a component containing a material capable of identifying an analyte or reacting with an analyte, the presence of which will be detected by an analyte sensor device. Typically, this material in the analyte sensing component usually produces a detectable signal via the electrode of the conductive component after interacting with the analyte to be sensed. In this regard, the electrodes of the analyte sensing component and the conductive component work in a combined manner to produce an electrical signal read by a device associated with the analyte sensor. Typically, the analyte sensing component includes an oxidoreductase, such as glucose oxidase, which is capable of reacting with a molecule and / or producing a molecule, the concentration change of which can be measured by measuring the current change at the electrode of the conductive component (e.g., oxygen and / or hydrogen peroxide). An enzyme capable of producing a molecule such as hydrogen peroxide can be arranged on an electrode according to many methods known in the art. The analyte sensing component can coat all or part of the various electrodes of the sensor. In this case, the analyte sensing component can coat the electrode to an equal degree. Alternatively, the analyte sensing component may coat different electrodes to different extents, such as the working electrode having a coated surface that is greater than the coated surface of the counter and / or reference electrodes.

[0086] Some sensor embodiments of this element of the invention utilize an enzyme (e.g., glucose oxidase) that has optionally been combined with a second protein (e.g., albumin) in a fixed ratio (e.g., a ratio typically optimized for the stability properties of glucose oxidase) and then applied to an electrode surface to form a thin enzyme component. In a typical embodiment, the analyte sensing component includes a GOx and HSA mixture. In a typical embodiment of an analyte sensing component with GOx, GOx reacts with glucose present in the sensing environment (e.g., the body of a mammal) and produces hydrogen peroxide.

[0087] As mentioned above, the enzyme and the second protein (e.g., albumin) can be treated to form a cross-linked matrix (e.g., by adding a cross-linking agent to the protein mixture). As known in the art, the cross-linking conditions can be controlled to adjust factors such as the retained biological activity of the enzyme, its mechanical and / or operational stability. Illustrative cross-linking procedures are described in U.S. Patent Application Serial No. 10 / 335,506 and PCT Publication WO 03 / 035891, which are incorporated herein by reference. For example, an amine cross-linking agent (such as, but not limited to, glutaraldehyde) can be added to the protein mixture (but in certain embodiments of the present invention disclosed herein, glutaraldehyde is excluded because adding a cross-linking agent to the protein mixture will produce a less active protein paste).

[0088] An alternative embodiment of the analyte sensing component is not formed using glutaraldehyde, but is formed to include a polypeptide that is encapsulated and / or cross-linked, such as glucose oxidase cross-linked with a polyvinyl alcohol (PVA, see, e.g., CAS No. 9002-89-5) polymer. As is known in the art, polyvinyl alcohol reacts with aldehydes to form water-insoluble polyacetals. In a pure PVA medium at a pH of about 5.0, the polymer reacts with the dialdehyde to form an acetal cross-linked structure. In certain embodiments of the invention, this cross-linking reaction can be carried out using a chemical vapor deposition (CVD) process. Due to the acidity of the PVA polymer solution, the cross-linking reaction in the CVD system is both simple and conventional. In addition, acidic conditions can be created by introducing compounds such as acetic acid into the glutaraldehyde solution, so the CVD system can provide acidic steam conditions. In addition, the pH of the polymer medium can be adjusted by adding acidic compounds such as citric acid, polymer additives such as polylysine, HBr, etc.

[0089] Embodiments of the analyte sensing component include compositions having properties that make them particularly suitable for use in mobile glucose sensors of the type worn by diabetic individuals. Such embodiments of the present invention include PVA-SbQ compositions for use in layered analyte sensor structures, which contain 1 mol% to 12.5 mol% SbQ. In certain embodiments of the present invention suitable for or used in glucose sensors, the ingredients in the layer are selected so that the molecular weight of the polyvinyl alcohol is between 30 kilodaltons and 150 kilodaltons, and the amount of SbQ present in the polyvinyl alcohol is between 1 mol% and 4 mol%. In some embodiments of the present invention, the analyte sensing layer is formed to contain from 5% to 12% PVA by weight. In some embodiments of the present invention, the analyte sensing layer is formed to contain glucose oxidase in an amount of 10 kU / ml to 20 kU / ml.

[0090] Embodiments of analyte sensing components include an analyte sensing layer, which is selected based on its ability to provide desired properties for implantable sensors. In certain embodiments of the present invention, the amount or proportion of PVA in the composition is used to adjust the water adsorption of the composition, the crosslinking density of the composition, etc. The effects of such preparations on phenomena such as H2O adsorption, sensor isig drift, and in vivo startup curves can be easily evaluated. Sufficient H2O adsorption can help maintain normal chemical and electrochemical reactions within amperometric analyte sensors. Therefore, it is desirable to form such a sensor from a composition with appropriate hydrophilic chemistry. In this case, the PVA-GOx composition disclosed herein can be used to produce an electrolyte hydrogel that can be used for an internal coating / membrane layer, and can also be coated on top of an analyte regulating layer (e.g., a glucose limiting membrane or "GLM") to improve the biocompatibility and hydrophilicity of the GLM layer.

[0091] As described above, in some embodiments of the present invention, the analyte sensing component includes a reagent (e.g., glucose oxidase) that can generate a signal (e.g., a change in oxygen and / or hydrogen peroxide concentration), which can be sensed by a conductive element (e.g., an electrode that senses a change in oxygen and / or hydrogen peroxide concentration). However, other useful analyte sensing components can be formed by any composition that can generate a detectable signal that can be sensed by a conductive element after interacting with a target analyte to be detected for its presence. In some embodiments, the composition includes an enzyme that regulates the concentration of hydrogen peroxide when reacting with the analyte to be sensed. Alternatively, the composition includes an enzyme that regulates the concentration of oxygen when reacting with the analyte to be sensed. In this case, a variety of enzymes that are used or produce hydrogen peroxide and / or oxygen in reactions with physiological analytes are known in the art, and these enzymes can be easily incorporated into the analyte sensing component composition. A variety of other enzymes known in the art can produce and / or utilize compounds that can be detected by a conductive element to regulate it, such as electrodes incorporated into the sensor design described herein. Such enzymes include, for example, those specifically described in Table 1, pages 15-29 and / or Table 18, pages 111-112 of Protein Immobilization: Fundamentals and Applications (Bioprocess Technology, Vol 14), Richard F. Taylor (ed.), Publisher: Marcel Dekker (January 7, 1991), the entire contents of which are incorporated herein by reference.

[0092] High density amine ingredients

[0093] The electrochemical sensor of the present invention comprises one or more high-density amine component layers (see, e.g. Figure 2BElement 500 in the sensor), which provides many beneficial functions for the sensor. Such a layer can optimize the sensor function, for example, by acting as an adhesion promoting component of a layer adjacent to the HDA layer, by reducing the fluctuations that may occur in a sensor based on glucose oxidase in the presence of oxygen concentration fluctuations, by improving the sensor initialization configuration, etc. The term "adhesion promoting component" is used herein according to professional terms recognized in the art, and refers to such a component, which contains a material selected for being able to promote adhesion between adjacent components in the sensor. Typically, the high-density amine adhesion promoting component is arranged between the analyte sensing component and the analyte regulating component, and is in direct contact with the analyte sensing component and the analyte regulating component. In a typical embodiment, the high-density amine layer 500 includes poly-L-lysine with a molecular weight between 30KDa and 300KDa (e.g., between 150KDa and 300KDa). In such a high-density amine layer 500, the concentration of poly-L-lysine is generally 0.1 wt% to 0.5 wt%, and the thickness of the high-density amine layer 500 is 0.1 to 0.4 microns. In an embodiment, the analyte sensing layer includes glucose oxidase so that the analyte sensor senses glucose, and the high-density amine layer 500 functions to reduce sensor signal variations due to fluctuations in oxygen (O 2 ) levels.

[0094] Analyte Modulating Components

[0095] The electrochemical sensors of the present invention include an analyte modulating component disposed on the sensor (see, e.g. Figure 2A 112 in). The term "analyte modulating component" is used herein according to professional terms recognized in the art, and refers to a component that typically forms a film on the sensor, which is used to regulate the diffusion of one or more analytes, such as glucose, through the component. In certain embodiments of the present invention, the analyte modulating component is an analyte limiting membrane, which is used to prevent or limit the diffusion of one or more analytes, such as glucose, through the component. In other embodiments of the present invention, the analyte modulating component is used to promote the diffusion of one or more analytes through the component. Alternatively, such an analyte modulating component can be formed to prevent or limit the diffusion of one type of molecule through the component (e.g., glucose), while allowing or even promoting the diffusion of other types of molecules through the component (e.g., O2).

[0096] About glucose sensor, in known enzyme electrode, glucose and oxygen from blood and some interferences such as ascorbic acid and uric acid are diffused through the primary membrane of sensor.When glucose, oxygen and interferences arrive at analyte sensing component, enzyme such as glucose oxidase catalyzes glucose to be converted into hydrogen peroxide and gluconolactone.Hydrogen peroxide can diffuse back through analyte regulating component, or can diffuse to electrode, where hydrogen peroxide can react to form oxygen and proton, thereby producing electric current proportional to glucose concentration.Analyte regulating sensor membrane assembly is used for several functions, including selectively allowing glucose to pass therethrough (see, e.g., U.S. Patent Application No. 2011-0152654).

[0097] C. Typical Analyte Sensor System Embodiments of the Invention

[0098] Embodiments of sensor elements and sensors can be operably coupled to a variety of other system elements (e.g., structural elements such as puncture members, insertion kits, etc., and electronic components such as processors, monitors, drug infusion pumps, etc.) typically used with analyte sensors, such as to make them suitable for use in various environments (e.g., implanted in mammals). One embodiment of the present invention includes a method for monitoring a user's physiological characteristics using an embodiment of the present invention, the embodiment including an input element capable of receiving a signal based on a sensed physiological characteristic value of the user from a sensor, and a processor for analyzing the received signal. In a typical embodiment of the present invention, the processor determines the dynamic behavior of the physiological characteristic value and provides an observable indicator based on the dynamic behavior of the physiological characteristic value so determined. In some embodiments, the physiological characteristic value is a measure of blood glucose concentration in the user. In other embodiments, the process of analyzing the received signal and determining the dynamic behavior includes repeatedly measuring the physiological characteristic value to obtain a series of physiological characteristic values, so as to, for example, incorporate comparative redundancy into the sensor device in a manner designed to provide confirmation information about sensor function, analyte concentration measurement, interference presence, etc.

[0099] Figure 4 Schematic diagram of a potentiostat that can be used to measure current in an embodiment of the present invention is shown. Figure 4 As shown, the potentiostat 300 may include an operational amplifier 310 connected in a circuit to have two inputs: Vset and Vmeasure. As shown, Vmeasure is a measurement of the voltage between the reference electrode and the working electrode. On the other hand, Vset is the optimal desired voltage between the working electrode and the reference electrode. The current between the counter electrode and the reference electrode is measured, thereby creating a current measurement result (isig) output from the potentiostat.

[0100] Embodiments of the present invention include such a device that processes display data from the measurement results of a sensed physiological characteristic (e.g., blood glucose concentration) in a customized manner and format to allow the user of the device to easily monitor and, if necessary, adjust the physiological state of the characteristic (e.g., regulate blood glucose concentration through insulin administration). An illustrative embodiment of the present invention is a device including the following: a sensor input terminal that is capable of receiving a signal from a sensor based on a sensed physiological characteristic value of a user; a memory that is used to store multiple measurement results of the sensed physiological characteristic value of the user based on the received signal from the sensor; and a display that is used to present a text and / or graphical representation of multiple measurement results of the sensed physiological characteristic value (e.g., text, line graph, etc., bar graph, etc., grid graph, etc., or a combination thereof). Typically, the graphical representation displays real-time measurement results of the sensed physiological characteristic value. Such a device can be used in a variety of situations, such as in combination with other medical devices. In some embodiments of the present invention, the device is used in combination with at least one other medical device (e.g., a glucose sensor).

[0101] The illustrative system embodiment is composed of a glucose sensor, a transmitter and a pump receiver and a glucose meter. In this system, a radio signal from a transmitter can be sent to a pump receiver every 5 minutes to provide a real-time sensor glucose (SG) value. The value / graph is displayed on the monitor of the pump receiver so that the user can monitor blood sugar by himself and use their own insulin pump to deliver insulin. Typically, the embodiment of the device disclosed herein communicates with a second medical device via a wired or wireless connection. Wireless communication may include, for example, receiving a radiation signal emitted, such as what occurs when a signal is transmitted by RF telemetry, infrared transmission, optical transmission, sound waves, and ultrasonic transmission. Optionally, the device is an integral part of a drug infusion pump (e.g., an insulin pump). Typically, in such a device, a physiological characteristic value includes a plurality of measurements of blood sugar.

[0102] Figure 3 A perspective view of a broad embodiment of a subcutaneous sensor insertion system and a block diagram of a sensor electronics device according to an illustrative embodiment of the present invention is provided. Additional elements commonly used with such sensor system embodiments are disclosed, for example, in U.S. Patent Application No. 20070163894, the contents of which are incorporated by reference. Figure 3A perspective view of a telemetric characteristic monitoring system 1 is provided, which includes a subcutaneous sensor kit 10 that is provided for subcutaneous placement of an active portion of a flexible sensor 12 or the like at a selected location on a user's body. The subcutaneous or percutaneous portion of the sensor kit 10 includes a hollow slotted insertion needle 14 having a sharp tip 44 and a cannula 16. Inside the cannula 16 is a sensing portion 18 of the sensor 12 to expose one or more sensor electrodes 20 to the user's bodily fluids through a window 22 formed in the cannula 16. The sensing portion 18 is joined to a connecting portion 24 that terminates in a conductive contact pad or the like, which is also exposed through one of the insulating layers. The connecting portion 24 and the contact pads are typically adapted for direct wired electrical connection to a suitable monitor 200 that is coupled to a display 214 to monitor a condition of the user in response to signals originating from the sensor electrodes 20. The connecting portion 24 can be conveniently electrically connected to the monitor 200 or the characteristic monitor transmitter 100 via a connector block 28 (or the like).

[0103] like Figure 3 As shown, according to an embodiment of the present invention, the subcutaneous sensor kit 10 can be configured or formed to work with a wired or wireless feature monitoring system. The proximal portion of the sensor 12 is mounted in a mounting base 30 suitable for placement on the user's skin. The mounting base 30 can be a pad having a lower surface, which is coated with a suitable pressure-sensitive adhesive layer 32. Before the sensor kit 10 is ready for use, a peeling paper tape 34 is usually provided to cover and protect the adhesive layer 32. The mounting base 30 includes an upper layer 36 and a lower layer 38, and the connecting portion 24 of the flexible sensor 12 is sandwiched between the layers 36 and 38. The connecting portion 24 has a front portion that is joined to the active sensing portion 18 of the sensor 12, and the front portion is folded at an angle to extend downward through a hole 40 formed in the lower base layer 38. Optionally, the adhesive layer 32 (or another part of the device that contacts the tissue in the body) includes an anti-inflammatory agent to reduce the inflammatory response and / or an antibacterial agent to reduce the chance of infection. The insertion needle 14 is adapted to be received by a needle port 42 formed in the upper base layer 36 and by a lower hole 40 in the lower base layer 38 in a sliding fit. After insertion, the insertion needle 14 is withdrawn to leave the cannula 16 with the sensing portion 18 and the sensor electrodes 20 at the selected insertion location. In this embodiment, the telemetry characteristic monitor transmitter 100 is coupled to the sensor kit 10 via a wire 102 through a connector 104, which is electrically coupled to the connector block 28 of the connector portion 24 of the sensor kit 10.

[0104] exist Figure 3In the illustrated embodiment, the telemetry characteristic monitor 100 includes a housing 106 that supports a printed circuit board 108, a battery 110, an antenna 112, and a wire 102 with a connector 104. In some embodiments, the housing 106 is formed of an upper housing 114 and a lower housing 116 that are ultrasonically welded to form a waterproof (or water-resistant) seal, thereby allowing cleaning by immersion (or wiping) with water, detergent, alcohol, etc. In some embodiments, the upper housing 114 and the lower housing 116 are made of medical grade plastic. However, in alternative embodiments, the upper housing 114 and the lower housing 116 can be connected together by other methods, such as snap fit, sealing rings, RTV (silicone sealant), and bonding together, etc., or formed of other materials, such as metals, composite materials, ceramics, etc. In other embodiments, a separate housing can be omitted and the assembly can be simply encapsulated in an epoxy or other moldable material that is compatible with the electronics and reasonably moisture-proof. As shown, the lower housing 116 may have an underside surface coated with a suitable pressure sensitive adhesive layer 118, with a release paper strip 120 typically provided to cover and protect the adhesive layer 118 until the sensor suite telemetry signature monitor transmitter 100 is ready for use.

[0105] exist Figure 3 In the illustrated illustrative embodiment, the subcutaneous sensor kit 10 facilitates the precise placement of a type of flexible thin film electrochemical sensor 12 for monitoring specific blood parameters indicative of a user's condition. The sensor 12 monitors glucose levels in the body and can be used in conjunction with an external or implantable automatic or semi-automatic drug infusion pump such as described in U.S. Pat. Nos. 4,562,751, 4,678,408, 4,685,903, or 4,573,994 to control insulin delivery to a diabetic patient.

[0106] exist Figure 3 In the illustrative embodiment shown in FIG. 1 , the sensor electrode 10 can be used in a variety of sensing applications and can be configured in a variety of ways. For example, the sensor electrode 10 can be used in physiological parameter sensing applications in which some types of biomolecules are used as catalysts. For example, the sensor electrode 10 can be used in glucose and oxygen sensors, which have glucose oxidase that catalyzes the reaction with the sensor electrode 20. The sensor electrode 10, along with the biomolecule or some other catalyst, can be placed in a vascular or non-vascular environment in the human body. For example, the sensor electrode 20 and the biomolecule can be placed in a vein and subjected to blood flow, or can be placed in the subcutaneous or peritoneal area of ​​the human body.

[0107] exist Figure 3In the illustrated embodiment of the invention, the monitor 200 of the sensor signal may also be referred to as the sensor electronics device 200. The monitor 200 may include a power supply, a sensor interface, processing electronics (i.e., a processor), and data formatting electronics. The monitor 200 may be coupled to the sensor kit 10 via a wire 102 through a connector that is electrically coupled to the connector block 28 of the connection portion 24. In an alternative embodiment, the wire may be omitted. In this embodiment of the invention, the monitor 200 may include a suitable connector for connecting directly to the connection portion 104 of the sensor kit 10. The sensor kit 10 may be modified so that the connector portion 104 is located in a different position, such as at the top of the sensor kit, to facilitate placing the monitor 200 above the sensor kit.

[0108] Although the analyte sensors and sensor systems disclosed herein are typically designed to be implanted in a mammal, the invention disclosed herein is not limited to any particular environment, but can be used in a variety of situations, such as for analyzing most in vivo and in vitro fluid samples, including biological fluids such as interstitial fluid, whole blood, lymph, plasma, serum, saliva, urine, feces, sweat, mucus, tears, cerebrospinal fluid, nasal secretions, cervical or vaginal secretions, semen, pleural fluid, amniotic fluid, peritoneal fluid, middle ear fluid, joint fluid, gastric fluid, etc. In addition, solid or dried samples can be dissolved in an appropriate solvent to provide a liquid mixture suitable for analysis.

[0109] It should be understood that the present invention is not limited to the specific embodiments described, as they can certainly vary. It should also be understood that the technical terms used herein are only for the purpose of describing specific embodiments, and are not intended to limit the present invention, as the scope of the present invention is limited only by the appended claims. In the description of the preferred embodiments, reference is made to the accompanying drawings that form a part thereof, and the accompanying drawings illustrate specific embodiments that can practice the present invention. It should be understood that other embodiments can be utilized and structural changes can be made without departing from the scope of the present invention.

Claims

1. An electrochemical analyte sensor comprising: Grassroots; A working electrode disposed on the substrate; and A multi-layer analyte sensor stack disposed on the working electrode, the multi-layer analyte sensor stack comprising: an analyte sensing layer disposed directly on the working electrode, wherein the analyte sensing layer detectably changes current at the working electrode in the presence of an analyte; a high-density amine layer disposed on the analyte sensing layer, wherein the high-density amine layer comprises a polymer having repeating amine groups; and an analyte modulating layer disposed on the high-density amine layer, wherein the analyte modulating layer modulates diffusion of analytes from an external environment to the working electrode; wherein: The high density amine layer includes a first side in direct contact with the analyte sensing layer and a second side in direct contact with the analyte modulating layer such that the high density amine layer bonds the analyte sensing layer to the analyte modulating layer.

2. The electrochemical analyte sensor according to claim 1, wherein The multi-layer analyte sensor stack does not include at least one of: another layer containing albumin; a further layer comprising a silicone adhesion promoter; or A layer comprising glutaraldehyde.

3. The electrochemical analyte sensor according to claim 1, wherein: The multi-layer analyte sensor stack consists essentially of (a)-(c).

4. The electrochemical analyte sensor according to claim 1, wherein: The high density amine layer serves as an adhesive layer that bonds the analyte sensing layer to the analyte modulating layer.

5. The electrochemical analyte sensor of claim 1, wherein: The high density amine layer includes a poly-L-lysine polymer having a molecular weight between 30 KDa and 300 KDa.

6. The electrochemical analyte sensor according to claim 5, wherein: The poly-L-lysine in the high-density amine layer has a molecular weight between 150 KDa and 300 KDa.

7. The electrochemical analyte sensor according to claim 5, wherein: The amount of poly-L-lysine in the high-density amine layer is 0.1 wt % to 0.5 wt %.

8. The electrochemical analyte sensor of claim 1, wherein: The thickness of the high-density amine layer is 0.1 to 0.4 microns.

9. The electrochemical analyte sensor of claim 1, wherein: The analyte sensing layer includes glucose oxidase disposed in the analyte sensing layer so that the analyte sensor senses glucose; and the high-density amine layer functions to reduce sensor signal variations caused by fluctuations in oxygen (O 2 ) levels.

10. A method of manufacturing an electrochemical analyte sensor, comprising the steps of: Disposing a working electrode on the substrate; disposing an analyte sensing layer on the working electrode, wherein the analyte sensing layer detectably changes the current at the working electrode in the presence of an analyte; directly disposing a high-density amine layer on the analyte sensing layer, the high-density amine layer comprising a polymer having repeating amine groups; and An analyte modulating layer is disposed directly on the high-density amine layer, wherein: The analyte modulating layer modulates diffusion of an analyte therethrough; and the high-density amine layer being formed to include a first side in direct contact with the analyte sensing layer and a second side in direct contact with the analyte modulating layer such that the high-density amine layer bonds the analyte sensing layer to the analyte modulating layer; Thus, an electrochemical analyte sensor is produced.

11. The method of claim 10, wherein the electrochemical analyte sensor comprises a multi-layer analyte sensor stack disposed on the working electrode, the multi-layer analyte sensor stack consisting essentially of the analyte sensor layer, the high-density amine layer, and the analyte modulating layer.

12. The method according to claim 11, wherein: The analyte sensing layer includes glucose oxidase disposed therein so that the analyte sensor senses glucose; and the high-density amine layer functions to reduce sensor signal variations due to fluctuations in oxygen (O2) levels during glucose sensing.

13. The method according to claim 10, wherein: The high density amine layer was deposited using a spray coating process and the sensor was exposed to an ethylene oxide sterilization process.

14. The method according to claim 13, wherein: The polymers having repeating amine groups include the following general structure: R 1 = Linear and / or branched alkyl functional groups of various chain lengths R 2 = Ketone functional group R 3 = Nitrogen functional group.

15. A method for sensing glucose concentration in a fluid, comprising: (a) placing an electrochemical glucose sensor in the fluid, wherein the electrochemical glucose sensor comprises: Grassroots; A working electrode disposed on the substrate; and A multi-layer analyte sensor stack, the multi-layer analyte sensor stack comprising: an analyte sensing layer comprising glucose oxidase disposed on the working electrode, wherein the analyte sensing layer detectably changes current at the working electrode in the presence of an analyte; a high-density amine layer comprising a poly-L-lysine polymer, wherein the high-density amine layer is disposed on the analyte sensing layer; and An analyte modulating layer disposed on the high-density amine layer, wherein: The analyte modulating layer modulates diffusion of glucose therethrough; and the high density amine layer comprising a first side in direct contact with the analyte sensing layer and a second side in direct contact with the analyte modulating layer such that the high density amine layer bonds the analyte sensing layer to the analyte modulating layer; (b) monitoring conductivity fluctuations; and (c) correlating said fluctuations in conductivity to glucose concentration; The glucose concentration in the fluid is thereby sensed.

16. The method of claim 15, wherein the fluid is interstitial fluid.

17. The method of claim 16, wherein the interstitial fluid is in an individual suffering from diabetes.

18. The method according to claim 17, wherein: The high density amine layer acts to reduce sensor signal variations due to fluctuations in oxygen (O 2 ) levels when sensing glucose concentration in the fluid.

19. The method according to claim 17, wherein: The analyte modulating layer comprises a polyurethane / polyurea polymer formed from a mixture comprising: (a) diisocyanates; (b) a hydrophilic polymer comprising a hydrophilic diol or a hydrophilic diamine; and (c) Siloxanes having an amino, hydroxyl or carboxylic acid functional group at the terminal.

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