Patent Publication Number: US-10772540-B2

Title: Microarray electrodes useful with analyte sensors and methods for making and using them

Description:
CROSS REFERENCE TO RELATED APPLICATIONS 
     This application is a divisional application which claims priority from U.S. patent application Ser. No. 13/707,400, filed Dec. 6, 2012, the contents of which are incorporated herein by reference. 
    
    
     TECHNICAL FIELD 
     The present invention relates to sensor electrodes useful in analyte sensor systems, such as glucose sensors used in the management of diabetes. 
     BACKGROUND OF THE INVENTION 
     Sensors are used to monitor a wide variety of compounds in various environments, including in vivo analytes. The quantitative determination of analytes in humans and mammals is of great importance in the diagnoses and maintenance of a number of pathological conditions. Illustrative analytes that are commonly monitored in a large number of individuals include glucose, lactate, cholesterol, and bilirubin. The determination of glucose concentrations in body fluids is of particular importance to diabetic individuals, individuals who must frequently check glucose levels in their body fluids to regulate the glucose intake in their diets. The results of such tests can be crucial in determining what, if any, insulin and/or other medication need to be administered. 
     Analyte sensors typically include components that convert interactions with analytes into detectable signals that can be correlated with the concentrations of the analyte. For example, some glucose sensors use amperometric means to monitor glucose in vivo. Such amperometric glucose sensors typically incorporate electrodes coated with glucose oxidase, an enzyme that catalyzes the reaction between glucose and oxygen to yield gluconic acid and hydrogen peroxide (H 2 O 2 ). The H 2 O 2  formed in this reaction alters an electrode current to form a detectable and measurable signal. Based on the signal, the concentration of glucose in the individual can then be measured. 
     An important component of analyte sensors and systems are the electrodes used to measure these signals. Based on various factors such as its design, plating, and the materials used, an electrode may vary in terms of its stability, reliability, and sensitivity in detecting analyte signals. In particular, electrode design plays an important role in the manufacturing of electrodes since it can determine how electric charges flow or build-up on electrodes. Generally, during the electroplating process, the intrinsic nature of electric charges is to accumulate at the edges of the electrode, especially at sharp edges and corners. This can create issues of edge growth and uneven plating thickness when the electrode is electroplated with an electrically conductive material. Furthermore, the non-uniform plating thickness often makes it harder to smoothly and uniformly dispose subsequent coatings and layers onto the electrode. 
     There is a need in the art for sensor electrode designs that can avoid undesirable issues associated edge growth. There is also a need for sensor electrodes and analyte sensor systems that have high stability, reliability, and sensitivity in detecting analyte signals. Embodiments of the invention disclosed herein meet these as well as other needs. 
     SUMMARY OF THE INVENTION 
     The invention disclosed herein includes microarray electrode designs as well as sensor apparatuses and systems that utilize such electrodes. As discussed below, working embodiments of the invention include electrodes formed from a plurality of electrically conductive elements, for example arrays of circular discs comprising electrodeposited platinum. The configuration of these elements and their associated electrode architectures facilitates an even distribution of electrical charges across the electrode, a phenomena that, for example, mitigates potentially problematic issues associated with charge accumulation. Such electrode designs provide a number of advantageous characteristics in certain contexts, for example by facilitating the detection of electrochemical reactions in devices such as amperometric glucose sensors. 
     The invention disclosed herein has a number of embodiments. One embodiment is a sensor electrode having an array of electroactive elements designed to enhance one or more functional characteristics of amperometric analyte sensors. Typically, the electrode comprises a base layer having a plurality of electrically conductive members formed on this layer and configured in an array. In such embodiments, the electrically conductive members comprise an electroactive surface adapted to sense fluctuations in electrical current at the electroactive surface; and the array of electrically conductive members is coupled to an electrical conduit. In typical embodiments of the invention, the electrode is operatively coupled to a processor adapted to store and/or process data obtained from observing fluctuations in electrical current. In some embodiments of the invention, the electrode is coupled to a piercing member adapted to be implanted in vivo. 
     In embodiments of the invention, the plurality of electrically conductive members are formed from shapes selected to avoid sharp edges and corners, electrode structures where electric charges can accumulate. In typical embodiments of the invention, the electrically conductive members can be formed to exhibit an ellipsoid geometry. For example, in some embodiments of the invention, the electrically conductive members comprise ellipses, circular discs, or combinations of ellipses and circular discs. Typically, such electrically conductive members are formed to have a diameter of at least 1 μm, for example, a diameter from 1 μm to 100 μm (e.g. circular discs having a diameter of 30, 40 or 50 μm). Optionally, the array comprises at least 5, 10, 20, 50 or 100 electrically conductive members. 
     Embodiments of the invention include analyte sensor apparatus designed to utilize the electrode microarrays disclosed herein. Such apparatuses typically include a base on which an array of electrically conductive members are disposed and configured to form a working electrode. Typically this base comprises a plurality of indentations (e.g. circular and/or elliptical indentations) and the plurality of electrically conductive members are individually positioned within the plurality of indentations. In some embodiments of the invention, the array of electrically conductive members is coupled to a common electrical conduit and a power source adapted to sense fluctuations in electrical current of the working electrode. 
     Typically, the electrode microarrays used in amperometric analyte sensing are coated with a plurality of materials having properties that, for example, facilitate analyte sensing. In some embodiments of the invention, an analyte sensing layer is disposed over the array of electrically conductive members, and includes an agent that is selected for its ability to detectably alter the electrical current at the working electrode in the presence of an analyte. In the working embodiments of the invention that are disclosed herein, the agent is glucose oxidase, a protein that undergoes a chemical reaction in the presence of glucose that results in an alteration in the electrical current at the working electrode. As disclosed below, these working embodiments can further include an analyte modulating layer disposed over the analyte sensing layer, wherein the analyte modulating layer modulates the diffusion of glucose as it migrates from an in vivo environment to the analyte sensing layer. In certain embodiments of the 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 silicone moiety. 
     Embodiments of the invention also include methods for making and using the electrode microarrays disclosed herein. For example another embodiment of the invention is a method of making a sensor electrode by obtaining or making a base structure having an outer surface, the outer surface comprising a plurality of circular indentations and then immersing the base in an electroplating electrolyte solution. In such methods, one can apply an electric field to solution in a manner that forms electrically conductive elements within the plurality of circular indentations (e.g. a plurality of circular discs formed from platinum black). In these methods, the array is constructed so that the plurality of conductive elements are connected to an electrical conduit and adapted to measure fluctuations in the array. 
     Yet another embodiment of the invention is a method of sensing an analyte within the body of a mammal. Typically this method comprises implanting an analyte sensor having an electrode microarray within the mammal (e.g. in the interstitial space of a diabetic individual), sensing an alteration in current at the working electrode in the presence of the analyte; and then correlating the alteration in current with the presence of the analyte, so that the analyte is sensed. While typical embodiments of the invention pertain to glucose sensors, the microarray electrode designs disclosed herein can be adapted for use with a wide variety of devices known in the art. 
     Other objects, features and advantages of the present invention will become apparent to those skilled in the art from the following detailed description. It is to be understood, however, that the detailed description and specific examples, while indicating some embodiments of the present invention are given by way of illustration and not limitation. Many changes and modifications within the scope of the present invention may be made without departing from the spirit thereof, and the invention includes all such modifications. 
    
    
     
       BRIEF DESCRIPTION OF THE DRAWINGS 
         FIGS. 1A-1B  illustrate: (A) a topographical view of an embodiment of a microarray sensor electrode; and (B) a close-up of a base layer and a plurality of circular discs on the sensor electrode. This illustrative embodiment comprises 119 micro discs having a diameter of 40 μm. The total array SA in this embodiment is equal to 0.001495 cm 2 . 
         FIG. 2  shows a sensor design comprising an amperometric analyte sensor formed from a plurality of planar layered elements. 
         FIG. 3  provides a perspective view illustrating a subcutaneous sensor insertion set, a telemetered characteristic monitor transmitter device, and a data receiving device embodying features of the invention. 
         FIGS. 4A-4D  illustrate various microarray electrode design embodiments of the invention.  FIG. 4A  shows an embodiment of the invention comprising 119 electrically conductive members (Pt discs in this embodiment) and designed so that the working electrode microarray has 38% the geometric surface area of a conventional working electrode and a working electrode to counter electrode ratio=&gt;1/5.  FIG. 4B  shows an embodiment of the invention comprising 200 electrically conductive members (Pt discs in this embodiment) and designed so that the working electrode microarray has 64% the geometric surface area of a conventional working electrode and a working electrode to counter electrode ratio=&gt;1/2.  FIG. 4C  shows an embodiment of the invention where both the working electrode and the counter electrode are formed from microarrays of electrically conductive members.  FIG. 4D  shows an embodiment of the invention comprising a plurality of working electrodes (comprising the array of electrically conductive members), counter electrodes and reference electrodes clustered together in units consisting essentially of one working electrode, one counter electrode and one reference electrode. 
         FIGS. 5A-5D  show:  FIG. 5(A)  a Boxplot graph of data showing SAR and plating conditions;  FIG. 5(B)  top (upper panel) and side (lower panel) view photographs of an electrical element comprising a circular disc formed from electrodeposited Pt;  FIG. 5(C)  angled (upper panel) and bird&#39;s eye (lower panel) view of an array comprising a circular discs formed by electrodepositing Pt in indentations in a base; and  FIG. 5(D)  a graph (upper panel) and graphic (lower panel) of data showing the cross section heights of circular discs formed by electrodepositing Pt, data that illustrates the relatively smooth topography of such rounded structures (relative to structures having well defined angles such as rectangles). 
         FIGS. 6A-6B  show: graphs illustrating  FIG. 6(A)  a COMSOL 4.2 simulation showing plating uniformity of an embodiment of a sensor electrode; and  FIG. 6(B)  two-dimensional plot of three-dimensional platinum electrode deposition thickness of a microarray of circular discs. COMSOL is a element analysis, solver and simulation software package physics and engineering applications. 
         FIGS. 7A-7C  provide:  FIG. 7(A)  and  FIG. 7(B)  data from studies of sensors having microarray electrodes in an in vitro testing system (SITS) that is designed to mimic in vivo conditions. In this system, sensor current is measured periodically in the presence of known concentrations of glucose and glucose values are then correlated with Isig, that is sensor current (in μA). These graphs provide data (Isig over periods of time) from experiments using sensors constructed to include different GLM analyte modulating compositions (A: blended polymers, B: a polyurethane/polyurea polymer) and electrode microarray formed from 40 μm platinum discs.  FIG. 7(C)  A Table summarizing the characteristics of these sensors and/or microarray electrodes. The data from these tests provides evidence that sensors formed with these microarray electrodes exhibit functional characteristics that are comparable, if not better than, sensors formed with conventional electrode designs. 
         FIGS. 8A-8C  provide:  FIG. 8(A)  and  FIG. 8(B)  graphs of in vivo data obtained from non-diabetic and diabetic dogs (sensor blood glucose mg/dL/sensor Isig-nA over 3 days of implantation using sensors constructed to include microarray electrodes). The graphs show that the sensor follows the in vivo glucose level changes very well in vivo from very beginning to the end of the implantation.  FIG. 8(C)  A Table summarizing the characteristics of these sensors and/or microarray electrodes. 
         FIGS. 9A-9B  show graphical data relating to glucose sensor responses to interfering species (e.g. acetaminophen).  FIG. 9A  shows a graph of in vivo data obtained from pigs with conventional sensors (lower panel) as well as sensors including microarray electrodes (upper panel). This data shows that sensors designed to include microarray electrodes exhibit a lower response to the interfering compound acetaminophen.  FIG. 9B  shows a graph of experimental data showing an in vitro interference response in microarray as compared to rectangular electrodes. This data shows that sensors with microarray electrodes exhibit a better (i.e. smaller percentage change in Isig) response to interferences than rectangular electrodes. 
         FIGS. 10A and 10B  provide graphs of data that illustrate sensor performance profiles that can be obtained with sensors constructed to include microarray electrodes. The graph in  FIG. 10A  provides data showing stable sensor Isig over 2 days of implantation in human body with s good in vivo sensor startup profile. Like the graph in  FIG. 10A , the graph in  FIG. 10B  shows very good low MARD (about 9%), indicating very limited deviation between sensed glucose and actual blood glucose. 
         FIG. 11  shows a schematic of a potentiostat that may be used to measure current in embodiments of the present invention. As shown in  FIG. 11 , a potentiostat  300  may include an op amp  310  that is connected in an electrical circuit so as to have two inputs: Vset and Vmeasured. As shown, Vmeasured is the measured value of the voltage between a reference electrode and a working electrode. Vset, on the other hand, is the optimally desired voltage across the working and reference electrodes. The current between the counter and reference electrode is measured, creating a current measurement (isig) that is output from the potentiostat. 
     
    
    
     DETAILED DESCRIPTION OF THE INVENTION 
     Unless otherwise defined, all terms of art, notations, and other scientific terms or terminology used herein are intended to have the meanings commonly understood by those of skill in the art to which this invention pertains. In some cases, terms with commonly understood meanings may be defined herein for clarity and/or for ready reference, and the inclusion of such definitions herein should not necessarily be construed to represent a substantial difference over what is generally understood in the art. Many of the techniques and procedures described or referenced herein are well understood and commonly employed using conventional methodology by those skilled in the art. As appropriate, procedures involving the use of commercially available kits and reagents are generally carried out in accordance with manufacturer defined protocols and/or parameters unless otherwise noted. A number of terms are defined below. 
     All numbers recited in the specification and associated claims that refer to values that can be numerically characterized with a value other than a whole number (e.g. the diameter of a circular disc) are understood to be modified by the term “about”. Where a range of values is provided, it is understood that each intervening value, to the tenth of the unit of the lower limit unless the context clearly dictates otherwise, between the upper and lower limit of that range and any other stated or intervening value in that stated range, is encompassed within the invention. The upper and lower limits of these smaller ranges may independently be included in the smaller ranges, and are also encompassed within the invention, subject to any specifically excluded limit in the stated range. Where the stated range includes one or both of the limits, ranges excluding either or both of those included limits are also included in the invention. Furthermore, all publications mentioned herein are incorporated herein by reference to disclose and describe the methods and/or materials in connection with which the publications are cited. Publications cited herein are cited for their disclosure prior to the filing date of the present application. Nothing here is to be construed as an admission that the inventors are not entitled to antedate the publications by virtue of an earlier priority date or prior date of invention. Further the actual publication dates may be different from those shown and require independent verification. 
     The term “analyte” as used herein is a broad term and is used in its ordinary sense, including, without limitation, to refer to a substance or chemical constituent in a fluid such as a biological fluid (for example, blood, interstitial fluid, cerebral spinal fluid, lymph fluid or urine) that can be analyzed. Analytes can include naturally occurring substances, artificial substances, metabolites, and/or reaction products. In common embodiments, the analyte is glucose. However, embodiments of the invention can be used with sensors designed for detecting a wide variety other analytes. Illustrative analytes include but are not limited to, lactate as well as salts, sugars, proteins fats, vitamins and hormones that naturally occur in vivo (e.g. in blood or interstitial fluids). The analyte can be naturally present in the biological fluid or endogenous; for example, a metabolic product, a hormone, an antigen, an antibody, and the like. Alternatively, the analyte can be introduced into the body or exogenous, for example, a contrast agent for imaging, a radioisotope, a chemical agent, a fluorocarbon-based synthetic blood, or a drug or pharmaceutical composition, including but not limited to insulin. The metabolic products of drugs and pharmaceutical compositions are also contemplated analytes. 
     The term “sensor” for example in “analyte sensor,” is used in its ordinary sense, including, without limitation, means used to detect a compound such as an analyte. A “sensor system” includes, for example, elements, structures and architectures (e.g. specific 3-dimensional constellations of elements) designed to facilitate sensor use and function. Sensor systems can include, for example, compositions such as those having selected material properties, as well as electronic components such as elements and devices used in signal detection and analysis (e.g. current detectors, monitors, processors and the like). 
     The terms “electrochemically reactive surface” and “electroactive surface” as used herein are broad terms and are used in their ordinary sense, including, without limitation, the surface of an electrode where an electrochemical reaction takes place. In one example, a working electrode measures hydrogen peroxide produced by the enzyme catalyzed reaction of the analyte being detected, creating an electric current (for example, detection of glucose analyte utilizing glucose oxidase produces H 2 O 2  as a byproduct, H 2 O 2  reacts with the surface of the working electrode producing two protons (2H + ), two electrons (2e − ) and one molecule of oxygen ( 02 ) which produces the electronic current being detected). In the case of the counter electrode, a reducible species, for example, O 2  is reduced at the electrode surface in order to balance the current being generated by the working electrode. 
     As discussed in detail below, embodiments of the invention relate to the use of an electrochemical sensor that measures a concentration of an analyte of interest or a substance indicative of the concentration or presence of the analyte in fluid. In some embodiments, the sensor is a continuous device, for example a subcutaneous, transdermal, or intravascular device. In some embodiments, the device can analyze a plurality of 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 indicative of the concentration of the analyte of interest. Typically, the sensor is of the type that senses a product or reactant of an enzymatic reaction between an analyte and an enzyme in the presence of oxygen as a measure of the analyte in vivo or in vitro. Such sensors typically comprise a membrane surrounding the enzyme through which an analyte migrates. The product is then measured using electrochemical methods and thus the output of an electrode system functions as a measure of the analyte. 
     Embodiments of the invention disclosed herein provide sensors of the type used, for example, in subcutaneous or transcutaneous monitoring of blood glucose levels in a diabetic patient. A variety of implantable, electrochemical biosensors have been developed for the treatment of diabetes and other life-threatening diseases. Many existing sensor designs use some form of immobilized enzyme to achieve their bio-specificity. Embodiments of the invention described herein can be adapted and implemented with a wide variety of known electrochemical sensors, including for example, U.S. Patent Application No. 20050115832, U.S. Pat. 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 as well as PCT International Publication Numbers WO 01/58348, WO 04/021877, WO 03/034902, WO 03/035117, WO 03/035891, WO 03/023388, WO 03/022128, WO 03/022352, WO 03/023708, WO 03/036255, WO03/036310 WO 08/042,625, and WO 03/074107, and European Patent Application EP 1153571, the contents of each of which are incorporated herein by reference. 
     Illustrative Embodiments of the Invention and Associated Characteristics 
     Embodiments of the invention disclosed herein provide sensors designed to include electrode microarrays that provide the sensors with enhanced functional and/or material properties. The disclosure further provides methods for making and using such sensors. As discussed in detail below, typical embodiments of the invention relate to the use of a sensor that measures a concentration of an aqueous analyte of interest or a substance indicative of the concentration or presence of the analyte in vivo. In some embodiments, the sensor is a subcutaneous, intramuscular, intraperitoneal, intravascular or transdermal device. Typically the sensor can be used for continuous analyte monitoring. In some embodiments, the device can analyze a plurality of 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 indicative of the concentration of the analyte of interest. 
     The invention disclosed herein has a number of embodiments. One embodiment is a sensor electrode having a unique configuration of electroactive elements. Typically, the electrode comprises a base layer having a plurality of electrically conductive members formed on this layer and configured in an array (see, e.g.  FIGS. 1 and 4 ). In such embodiments, the electrically conductive members comprise an electroactive surface adapted to sense fluctuations in electrical current at the electroactive surface; and the array of electrically conductive members is coupled to an electrical conduit. 
     Embodiments of the invention exhibit a number of advantages over conventional electrode designs. For example, during the electroplating process, the intrinsic nature of electric charges is to accumulate at the edges of electrodes, especially at sharp edges and corners, which result in edge growth and uneven electrodeposition on the surface of the electrode, features which can introduce problematical phenomena in certain electrode contexts. Embodiments of the present invention organize an electrode into small micro-arrays of circular discs. The plurality of circular discs allows the charges to evenly distribute around the discs, thereby mitigating charge accumulation along the edges of the electrode. This eliminates or drastically reduces edge growth on the electrode and allows for more uniform plating during the electroplating process. In this context, electroplating uniformity plays an important role in the reliability of the sensor electrodes. An improved electroplating uniformity of platinum in certain embodiments of the present invention provided good stability, linearity, and glucose tracking in vivo. Additionally, the electroplating uniformity provided with the methods disclosed herein benefit any subsequent layer coatings disposed over the electrode by providing a smoother surface on which to dispose coating, thereby facilitating layer coating adherence and lamination. 
     Embodiments of the invention are designed to improve electroplating uniformity of platinum and the mitigation/elimination of edge growth on sensor electrodes. Specifically, electrode design can play an important role in electroplating processes as it can determine how electric charges would flow or build-up on the electrodes. In particular, during the electroplating process, the intrinsic nature of electric charges is to accumulate at the edges of electrodes, specifically at sharp edges and corners. In this context, embodiments of the invention provide a beneficial function by: (1) having a electrically conductive element shape (e.g. circular discs); in combination with (2) breaking down the electrode into small micro-arrays, characteristics selected so that the charges will evenly distribute around the microarrays circles, thereby mitigating charge accumulation and resulting in less edge growth and more uniform plating. 
     As disclosed herein, the disclosed electrode microarrays provide electrodes and sensors having enhanced functional and/or material properties. Such advantages can include, for example, a more uniformly electroplated surface as well as more sites for electrochemical reactions, features which contribute to the stability and/or sensitivity of layered sensor structures. For example, as shown in  FIGS. 5-10 , the selected architectures and configurations of elements used in embodiments of the invention provide a better uniformity for electroplating, and therefore less edge growth. In addition to helping to avoid charge accumulation, this reduction in edge growth can facilitate the production of sensors where the electrodes are coated by layers of material layer coating, specifically by providing a “smoother” surface that can contribute to sensor stability by decreasing the possibility that one or more layers of material may delaminate. Sensor embodiments of the invention that include a base comprising a plurality of indentations in which the plurality of electrically conductive members are individually disposed within the plurality of indentations (see, e.g.  FIG. 5 ) further contribute to sensor stability because: (1) a higher ratio of polyimide to electroplated metals provides an ideal surface for better adhesion of layered materials coated on the electrodes; and (2) a higher ratio of base material (e.g. polyimide) around the electrodes helps to secure/lock-down metal layer to sensor base. 
     Embodiments of the invention can also facilitate the sensitivity of certain electrochemical sensor designs because, for example, the perimeter (contour) of the plurality of circular discs is greater than the perimeter of conventional sensor electrodes and thus provides more sites for electrochemical reactions. Embodiments of the invention having a plurality of circular discs of electrically conductive elements exhibit a radial diffusion effect, a phenomena which enhances the mass transport of analytes to the electrode interfaces, thereby enhancing sensor sensitivity (see  FIG. 4 ). In addition, as shown in  FIG. 9 , the effects of interferants on the signals of electrochemical glucose sensors having either microarray electrodes formed from an array of circular Pt discs (as electrically conductive elements) or conventional rectangular electrodes were tested in an in vivo model. As shown in  FIG. 9 , glucose sensors having microarray electrodes exhibit a reduced interference response. 
     In typical embodiments of the invention, electrochemical sensors are operatively coupled to a sensor input capable of receiving signals 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. Optionally, a pulsed voltage is used to obtain a signal from an electrode. In certain embodiments of the invention, the processor is capable of comparing a first signal received from a working electrode in response to a first working potential with a second signal received from a working electrode in response to a second working potential. Optionally, the electrode is coupled to a processor adapted to convert data obtained from observing fluctuations in electrical current from a first format into a second format. Such embodiments include, for example, processors designed to convert a sensor current Input Signal (e.g. ISIG measured in nA) to a blood glucose concentration. 
     In many embodiments of the invention, the sensors comprise a biocompatible region adapted to be implanted in vivo. In some embodiments, the sensor comprises a discreet probe that pierces an in vivo environment. In embodiments of the invention, the biocompatible region can comprise a polymer that contacts an in vivo tissue. Optionally, the polymer is a hydrophilic polymer (e.g. one that absorbs water). In this way, sensors used in the systems of the invention can be used to sense a wide variety of analytes in different aqueous environments. In some embodiments of the invention, the electrode is coupled to a piercing member (e.g. a needle) adapted to be implanted in vivo. While sensor embodiments of the invention can comprise one or two piercing members, optionally such sensor apparatuses can include 3 or 4 or 5 or more piercing members that are coupled to and extend from a base element and are operatively coupled to 3 or 4 or 5 or more electrochemical sensors (e.g. microneedle arrays, embodiments of which are disclosed for example in U.S. Pat. Nos. 7,291,497 and 7,027,478, and U.S. patent Application No. 20080015494, the contents of which are incorporated by reference). 
     In embodiments of the invention, the plurality of electrically conductive members are formed from shapes selected to avoid sharp edges and corners, electrode structures where electric charges can accumulate. In common embodiments of the invention, the base comprises a plurality of indentations and the plurality of electrically conductive members are individually positioned within the plurality of indentations. In typical embodiments of the invention, the electrically conductive members can be formed to exhibit an ellipsoid geometry. For example, in some embodiments of the invention, the electrically conductive members comprise ellipses or circular discs. In some embodiments of the invention, the electrically conductive members comprise combinations of ellipses and circular discs. Typically, such electrically conductive members are formed to have a diameter of at least 1 μm, for example, a diameter from 1 μm to 100 μm (e.g. a diameter of about 10-50 or 20-40 μm). In certain embodiments of the invention, the electrically conductive members comprise elliptical shapes (e.g. circular discs) and have a diameter of at least 10, 20, 30, 40, 50, 60, 70, 80 or 90 μm. Optionally, the array comprises at least 5, 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, 125, 150 175 or 200 electrically conductive members. 
     In embodiments of the invention, the microarray electrodes can function as individually addressable working electrodes. Embodiments of the invention include analyte sensor apparatus designed to utilize the electrodes disclosed herein. Such apparatuses typically include a base on which an array of electrically conductive members are disposed and configured to form a working electrode. Typically this base comprises a plurality of indentations and the plurality of electrically conductive members are individually positioned within the plurality of indentations and the electrically conductive members comprise an electroactive surface adapted to sense fluctuations in electrical current at the electroactive surface. In typical embodiments of the invention, one or more electrically conductive elements can be connected using conventional elements such as ones or more vertical electrical connections between layers of conductive elements in an electronic circuit. In some embodiments of the invention, the array of electrically conductive members is coupled to a common electrical element/conduit (e.g. so that the conductive members of the array are not separately wired, and are instead electrically linked as a group). In other embodiments of the invention, one or more of the electrically conductive members in the array (e.g. at least 1, 5, 10, 25, 50, 100 or all) of the electrically conductive members in the array) is coupled to an individual electrical element/conduit (e.g. so that these one or more conductive member(s) in the array is/are separately wired). 
     Optionally, the electrical conduit is coupled to a power source adapted to sense fluctuations in electrical current of the array of the working electrode. Typically the apparatus include a reference electrode; and a counter electrode. Optionally one or more of these electrodes also comprises a plurality of electrically conductive members disposed on the base in an array. In some embodiments, each of the electrically conductive members of the electrode (e.g. the counter electrode) comprises an electroactive surface adapted to sense fluctuations in electrical current at the electroactive surface; and the group of electrically conductive members are coupled to a power source (e.g. a potentiostat or the like). 
     In some embodiments of the invention, the apparatus comprises a plurality of working electrodes, counter electrodes and reference electrodes clustered together in units consisting essentially of one working electrode, one counter electrode and one reference electrode; and the clustered units are longitudinally distributed on the base layer in a repeating pattern of units (see, e.g.  FIG. 4D ). In some sensor embodiments, the distributed electrodes are organized/disposed within a flex-circuit assembly (i.e. a circuitry assembly that utilizes flexible rather than rigid materials). Such flex-circuit assembly embodiments provide an interconnected assembly of elements (e.g. electrodes, electrical conduits, contact pads and the like) configured to facilitate wearer comfort (for example by reducing pad stiffness and wearer discomfort). 
     Typically, the sensor electrode microarrays of the invention are coated with a plurality of materials having properties that, for example, facilitate analyte sensing. In some embodiments of the invention, an analyte sensing layer is disposed over the array of electrically conductive members, and includes an agent that is selected for its ability to detectably alter the electrical current at the working electrode in the presence of an analyte. In the working embodiments of the invention that are disclosed herein, the agent is glucose oxidase, a protein that undergoes a chemical reaction in the presence of glucose that results in an alteration in the electrical current at the working electrode. These working embodiments further include an analyte modulating layer disposed over the analyte sensing layer, wherein the analyte modulating layer modulates the diffusion of glucose as it migrates from an in vivo environment to the analyte sensing layer. In certain embodiments of the 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 silicone moiety. In certain embodiments of the 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 at a ratio of between 1:1 and 1:20 (e.g. 1:2) by weight %. In working embodiments of the present invention, the signal strength and O 2  response of the microarray sensor electrode can be increased with the use of a 2× permselective GLM (glucose limiting membrane). Typically, this analyte modulating layer composition comprises a first polymer formed from a mixture comprising a diisocyanate; at least one hydrophilic diol or hydrophilic diamine; and a siloxane; that is blended with a second polymer formed from a mixture comprising: a 2-(dimethylamino)ethyl methacrylate; a methyl methacrylate; a polydimethyl siloxane monomethacryloxypropyl; a poly(ethylene oxide) methyl ether methacrylate; and a 2-hydroxyethyl methacrylate. Additional material layers can be included in such apparatuses. For example, in some embodiments of the invention, the apparatus comprises an adhesion promoting layer disposed between the analyte sensing layer and the analyte modulating layer. 
     One sensor embodiment shown in  FIG. 2  is a amperometric sensor  100  having a plurality of layered elements including a base layer  102 , a conductive layer  104  (e.g. one comprising the plurality of electrically conductive members) which is disposed on and/or combined with the base layer  102 . Typically the conductive layer  104  comprises one or more electrodes. An analyte sensing layer  110  (typically comprising an enzyme such as glucose oxidase) is disposed on one or more of the exposed electrodes of the conductive layer  104 . A protein layer  116  disposed upon the analyte sensing layer  110 . An analyte modulating layer  112  is disposed above the analyte sensing layer  110  to regulate analyte (e.g. glucose) access with the analyte sensing layer  110 . An adhesion promoter layer  114  is disposed between layers such as the analyte modulating layer  112  and the analyte sensing layer  110  as shown in  FIG. 2  in order to facilitate their contact and/or adhesion. This embodiment also comprises a cover layer  106  such as a polymer coating can be disposed on portions of the sensor  100 . Apertures  108  can be formed in one or more layers of such sensors. Amperometric glucose sensors having 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. 
     Embodiments of the invention also include methods for making and using the electrode microarrays disclosed herein. For example yet another embodiment of the invention is a method of making a sensor electrode by obtaining or making a base structure having an outer surface, the outer surface comprising a plurality of circular indentations and then immersing the base in an electroplating electrolyte solution. In embodiments of the invention, the substrate on which the metal is electrodeposited can comprise various conventional substrates made from different materials such as polymers, metals and the like. In some embodiments of the invention, the substrate on which the metal is electrodeposited can comprise patterned metal. In typical embodiments of the invention, one or more electrically conductive elements can be connected using conventional elements such as ones or more vias (Vertical Interconnect Access), elements that comprise vertical electrical connections between different layers of conductors in a physical electronic circuit. In such methods, one can apply an electric field to solution in a manner that forms electrically conductive elements within the plurality of circular or elliptical indentations (e.g. a plurality of circular discs formed from platinum black). In these methods, the array is constructed so that each of the plurality of conductive elements are connected to a shared electrical conduit such that the conduit can be used to obtain useful information on the electrical profile of the whole array (e.g. information on electrical current fluctuations in the aggregated conductive elements). 
     Yet another embodiment of the invention is a method of sensing an analyte within the body of a mammal. Typically this method comprises implanting an analyte sensor having an electrode microarray within the mammal (e.g. in the interstitial space of a diabetic individual), sensing an alteration in current at the working electrode in the presence of the analyte; and then correlating the alteration in current with the presence of the analyte, so that the analyte is sensed. 
     Embodiments of the invention also provide articles of manufacture and kits for observing a concentration of an analyte. In an illustrative embodiment, the kit includes a sensor comprising electrode microarrays as discussed herein. In typical embodiments, the sensors are disposed in the kit within a sealed sterile dry package. Optionally the kit comprises an insertion device that facilitates insertion of the sensor. The kit and/or sensor set typically comprises a container, a label and an analyte sensor as described above. Suitable containers include, for example, an easy to open package made from a material such as a metal foil, bottles, vials, syringes, and test tubes. The containers may be formed from a variety of materials such as metals (e.g. foils) paper products, glass or plastic. The label on, or associated with, the container indicates that the sensor is used for assaying the analyte of choice. The kit and/or sensor set may include other materials desirable from a commercial and user standpoint, including buffers, diluents, filters, needles, syringes, and package inserts with instructions for use. 
     Specific aspects of embodiments of the invention are discussed in detail in the following sections. 
     Typical Elements, Configurations and Analyte Sensor Embodiments of the Invention 
     A. Typical Elements Found in of Embodiments of the Invention 
       FIGS. 1 and 4  provide illustrations of various microarray electrode design embodiments of the invention. 
       FIG. 2  illustrates a cross-section of a typical sensor embodiment  100  of the present invention. This sensor embodiment is formed from a plurality of components that are typically in the form of layers of various conductive and non-conductive constituents disposed on each other according to art accepted methods and/or the specific methods of the invention disclosed herein. The components of the sensor are typically characterized herein as layers because, for example, it allows for a facile characterization of the sensor structure shown in  FIG. 2 . Artisans will understand however, that in certain embodiments of the invention, the sensor constituents are combined such that multiple constituents form one or more heterogeneous layers. In this context, those of skill in the art understand that the ordering of the layered constituents can be altered in various embodiments of the invention. 
     The embodiment shown in  FIG. 2  includes a base layer  102  to support the sensor  100 . The base layer  102  can be made of a material such as a metal and/or a ceramic and/or a polymeric substrate, which may be self-supporting or further supported by another material as is known in the art. Embodiments of the invention include a conductive layer  104  which is disposed on and/or combined with the base layer  102 . Typically the conductive layer  104  comprises one or more electrically conductive elements that function as electrodes. An 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 and/or counter and/or reference electrodes and/or one or more electrodes that performs multiple functions, for example one that functions as both as a reference and a counter electrode. 
     As discussed in detail below, the base layer  102  and/or conductive layer  104  can be generated using many known techniques and materials. In certain embodiments of the invention, the electrical circuit of the sensor is defined by etching the disposed conductive layer  104  into a desired pattern of conductive paths. A typical electrical circuit for the sensor  100  comprises two or more adjacent conductive paths with regions at a proximal end to form contact pads and regions at a distal end to form sensor electrodes. An electrically insulating cover layer  106  such as a polymer coating can be disposed on portions of the sensor  100 . Acceptable polymer coatings for use as the 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, or the like. In the sensors of the present invention, one or more exposed regions or apertures  108  can be made through the cover layer  106  to open the conductive layer  104  to the external environment and to, for example, allow an analyte such as glucose to permeate the layers of the sensor and be sensed by the sensing elements. Apertures  108  can be formed by a number of techniques, including laser ablation, tape masking, chemical milling or etching or photolithographic development or the like. In certain embodiments of the invention, during manufacture, a secondary photoresist can also be applied to the protective layer  106  to define the regions of the protective layer to be removed to form the aperture(s)  108 . The exposed electrodes and/or contact pads can also undergo secondary processing (e.g. through the apertures  108 ), such as additional plating processing, to prepare the surfaces and/or strengthen the conductive regions. 
     In the sensor configuration shown in  FIG. 2 , an analyte sensing layer  110  is disposed on one or more of the exposed electrodes of the conductive layer  104 . Typically, the analyte sensing layer  110  is an enzyme layer. Most typically, the analyte sensing layer  110  comprises an enzyme capable of producing and/or utilizing oxygen and/or hydrogen peroxide, for example the enzyme 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 or the like. In an illustrative embodiment, an oxidoreductase enzyme such as glucose oxidase in the analyte sensing layer  110  reacts with glucose to produce hydrogen peroxide, a compound which then modulates a current at an electrode. As this modulation of current depends on the concentration of hydrogen peroxide, and the concentration of hydrogen peroxide correlates to the concentration of glucose, the concentration of glucose can be determined by monitoring this modulation in the current. In a specific embodiment of the invention, the hydrogen peroxide is oxidized at a working electrode which is an anode (also termed herein the anodic working electrode), with the resulting current being proportional to the hydrogen peroxide concentration. Such modulations in the current caused by changing hydrogen peroxide concentrations can by monitored by any one of a variety of sensor detector apparatuses such as a universal sensor amperometric biosensor detector or one of the other variety of similar devices known in the art such as glucose monitoring devices produced by Medtronic Diabetes. 
     In embodiments of the invention, the analyte sensing layer  110  can be applied over portions of the conductive layer or over the entire region of the conductive layer. Typically the analyte sensing layer  110  is disposed on the working electrode which can be the anode or the cathode. Optionally, the analyte sensing layer  110  is also disposed on a counter and/or reference electrode. Methods for generating a thin analyte sensing layer  110  include brushing the layer onto a substrate (e.g. the reactive surface of a platinum black electrode), as well as spin coating processes, dip and dry processes, low shear spraying processes, ink-jet printing processes, silk screen processes and the like. In certain embodiments of the invention, brushing is used to: (1) allow for a precise localization of the layer; and (2) push the layer deep into the architecture of the reactive surface of an electrode (e.g. platinum black produced by an electrodeposition process). 
     Typically, the analyte sensing layer  110  is coated and or disposed next to one or more additional layers. Optionally, the one or more additional layers includes a protein layer  116  disposed upon the analyte sensing layer  110 . Typically, the protein layer  116  comprises a protein such as human serum albumin, bovine serum albumin or the like. Typically, the protein layer  116  comprises human serum albumin. In some embodiments of the invention, an additional layer includes an analyte modulating layer  112  that is disposed above the analyte sensing layer  110  to regulate analyte contact with the analyte sensing layer  110 . For example, the analyte modulating membrane layer  112  can comprise a glucose limiting membrane, which regulates the amount of glucose that contacts an enzyme such as glucose oxidase that is present in the analyte sensing layer. Such glucose limiting membranes can be made from a wide variety of materials known to be suitable for such purposes, e.g., silicone compounds such as polydimethyl siloxanes, polyurethanes, polyurea cellulose acetates, Nafion, polyester sulfonic acids (e.g. Kodak AQ), hydrogels or any other suitable hydrophilic membranes known to those skilled in the art. 
     In typical embodiments of the invention, an adhesion promoter layer  114  is disposed between the analyte modulating layer  112  and the analyte sensing layer  110  as shown in  FIG. 2  in order to facilitate their contact and/or adhesion. In a specific embodiment of the invention, an adhesion promoter layer  114  is disposed between the analyte modulating layer  112  and the protein layer  116  as shown in  FIG. 2  in order to facilitate their contact and/or adhesion. The adhesion promoter layer  114  can be made from any one of a wide variety of materials known in the art to facilitate the bonding between such layers. Typically, the adhesion promoter layer  114  comprises a silane compound. In alternative embodiments, protein or like molecules in the analyte sensing layer  110  can be sufficiently crosslinked or otherwise prepared to allow the analyte modulating membrane layer  112  to be disposed in direct contact with the analyte sensing layer  110  in the absence of an adhesion promoter layer  114 . 
     B. Typical Analyte Sensor Constituents Used in Embodiments of the Invention 
     The following disclosure provides examples of typical elements/constituents used in sensor embodiments of the invention. While these elements can be described as discreet units (e.g. layers), those of skill in the art understand that sensors can be designed to contain elements having a combination of some or all of the material properties and/or functions of the elements/constituents discussed below (e.g. an element that serves both as a supporting base constituent and/or a conductive constituent and/or a matrix for the analyte sensing constituent and which further functions as an electrode in the sensor). Those in the art understand that these thin film analyte sensors can be adapted for use in a number of sensor systems such as those described below. 
     Base Constituent 
     Sensors of the invention typically include a base constituent (see, e.g. element  102  in  FIG. 2 ). The term “base constituent” is used herein according to art accepted terminology and refers to the constituent in the apparatus that typically provides a supporting matrix for the plurality of constituents that are stacked on top of one another and comprise the functioning sensor. In one form, the base constituent comprises a thin film sheet of insulative (e.g. electrically insulative and/or water impermeable) material. This base constituent can be made of a wide variety of materials having desirable qualities such as dielectric properties, water impermeability and hermeticity. Some materials include metallic, and/or ceramic and/or polymeric substrates or the like. 
     Conductive Constituent 
     The electrochemical sensors of the invention typically include a conductive constituent disposed upon the base constituent that includes at least one electrode for contacting an analyte or its byproduct (e.g. oxygen and/or hydrogen peroxide) to be assayed (see, e.g. element  104  in  FIG. 2 ). The term “conductive constituent” is used herein according to art accepted terminology and refers to electrically conductive sensor elements such as a plurality of electrically conductive members disposed on the base layer in an array (e.g. so as to form a microarray electrode) and which are capable of measuring a detectable signal and conducting this to a detection apparatus. An illustrative example of this is a conductive constituent that forms a working electrode that can measure an increase or decrease in current in response to exposure to a stimuli such as the change in the concentration of an analyte or its byproduct as compared to a reference electrode that does not experience the change in the concentration of the analyte, a coreactant (e.g. oxygen) used when the analyte interacts with a composition (e.g. the enzyme glucose oxidase) present in analyte sensing constituent  110  or a reaction product of this interaction (e.g. hydrogen peroxide). Illustrative examples of such elements include electrodes which are capable of producing variable detectable signals in the presence of variable concentrations of molecules such as hydrogen peroxide or oxygen. 
     In addition to the working electrode, the analyte sensors of the invention typically include a reference electrode or a combined reference and counter electrode (also termed a quasi-reference electrode or a counter/reference electrode). If the sensor does not have a counter/reference electrode then it may include a separate counter electrode, which may be made from the same or different materials as the working electrode. Typical sensors of the present invention have one or more working electrodes and one or more counter, reference, and/or counter/reference electrodes. One 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 integrally connected or they may be kept separate. Optionally, the electrodes can be disposed on a single surface or side of the sensor structure. Alternatively, the electrodes can be disposed on a multiple surfaces or sides of the sensor structure (and can for example be connected by vias through the sensor material(s) to the surfaces on which the electrodes are disposed). In certain embodiments of the invention, the reactive surfaces of the electrodes are of different relative areas/sizes, for example a 1× reference electrode, a 2.6× working electrode and a 3.6× counter electrode. 
     Interference Rejection Constituent 
     The electrochemical sensors of the invention optionally include an interference rejection constituent disposed between the surface of the electrode and the environment to be assayed. In particular, certain sensor embodiments rely on the oxidation and/or reduction of hydrogen peroxide generated by enzymatic reactions on the surface of a working electrode at a constant potential applied. Because amperometric detection based on direct oxidation of hydrogen peroxide requires a relatively high oxidation potential, sensors employing this detection scheme may suffer interference from oxidizable species that are present in biological fluids such as ascorbic acid, uric acid and acetaminophen. In this context, the term “interference rejection constituent” is used herein according to art accepted terminology and refers to a coating or membrane in the sensor that functions to inhibit spurious signals generated by such oxidizable species which interfere with the detection of the signal generated by the analyte to be sensed. Certain interference rejection constituents function via size exclusion (e.g. by excluding interfering species of a specific size). Examples of interference rejection constituents include one or more layers or coatings of compounds such as hydrophilic polyurethanes, cellulose acetate (including cellulose acetate incorporating agents such as poly(ethylene glycol), polyethersulfones, polytetra-fluoroethylenes, the perfluoronated ionomer Nafion™, polyphenylenediamine, epoxy and the like. 
     Analyte Sensing Constituent 
     The electrochemical sensors of the invention include an analyte sensing constituent disposed on the electrodes of the sensor (see, e.g. element  110  in  FIG. 2 ). The term “analyte sensing constituent” is used herein according to art accepted terminology and refers to a constituent comprising a material that is capable of recognizing or reacting with an analyte whose presence is to be detected by the analyte sensor apparatus. Typically this material in the analyte sensing constituent produces a detectable signal after interacting with the analyte to be sensed, typically via the electrodes of the conductive constituent. In this regard the analyte sensing constituent and the electrodes of the conductive constituent work in combination to produce the electrical signal that is read by an apparatus associated with the analyte sensor. Typically, the analyte sensing constituent comprises an oxidoreductase enzyme capable of reacting with and/or producing a molecule whose change in concentration can be measured by measuring the change in the current at an electrode of the conductive constituent (e.g. oxygen and/or hydrogen peroxide), for example the enzyme glucose oxidase. An enzyme capable of producing a molecule such as hydrogen peroxide can be disposed on the electrodes according to a number of processes known in the art. The analyte sensing constituent can coat all or a portion of the various electrodes of the sensor. In this context, the analyte sensing constituent may coat the electrodes to an equivalent degree. Alternatively the analyte sensing constituent may coat different electrodes to different degrees, with for example the coated surface of the working electrode being larger than the coated surface of the counter and/or reference electrode. 
     Typical sensor embodiments of this element of the invention utilize an enzyme (e.g. glucose oxidase) that has been combined with a second protein (e.g. albumin) in a fixed ratio (e.g. one that is typically optimized for glucose oxidase stabilizing properties) and then applied on the surface of an electrode to form a thin enzyme constituent. In a typical embodiment, the analyte sensing constituent comprises a GOx and HSA mixture. In a typical embodiment of an analyte sensing constituent having GOx, the GOx reacts with glucose present in the sensing environment (e.g. the body of a mammal) and generates hydrogen peroxide. 
     As noted above, the enzyme and the second protein (e.g. an albumin) are typically treated to form a crosslinked matrix (e.g. by adding a cross-linking agent to the protein mixture). As is known in the art, crosslinking conditions may be manipulated to modulate factors such as the retained biological activity of the enzyme, its mechanical and/or operational stability. Illustrative crosslinking procedures are described in U.S. patent application Ser. No. 10/335,506 and PCT publication WO 03/035891 which are incorporated herein by reference. For example, an amine cross-linking reagent, such as, but not limited to, glutaraldehyde, can be added to the protein mixture. The addition of a cross-linking reagent to the protein mixture creates a protein paste. The concentration of the cross-linking reagent to be added may vary according to the concentration of the protein mixture. While glutaraldehyde is an illustrative crosslinking reagent, other cross-linking reagents may also be used or may be used in place of glutaraldehyde. Other suitable cross-linkers also may be used, as will be evident to those skilled in the art. 
     As noted above, in some embodiments of the invention, the analyte sensing constituent includes an agent (e.g. glucose oxidase) capable of producing a signal (e.g. a change in oxygen and/or hydrogen peroxide concentrations) that can be sensed by the electrically conductive elements (e.g. electrodes which sense changes in oxygen and/or hydrogen peroxide concentrations). However, other useful analyte sensing constituents can be formed from any composition that is capable of producing a detectable signal that can be sensed by the electrically conductive elements after interacting with a target analyte whose presence is to be detected. In some embodiments, the composition comprises an enzyme that modulates hydrogen peroxide concentrations upon reaction with an analyte to be sensed. Alternatively, the composition comprises an enzyme that modulates oxygen concentrations upon reaction with an analyte to be sensed. In this context, a wide variety of enzymes that either use or produce hydrogen peroxide and/or oxygen in a reaction with a physiological analyte are known in the art and these enzymes can be readily incorporated into the analyte sensing constituent composition. A variety of other enzymes known in the art can produce and/or utilize compounds whose modulation can be detected by electrically conductive elements such as the electrodes that are incorporated into the sensor designs described herein. Such enzymes include for example, enzymes 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) by Richard F. Taylor (Editor) Publisher: Marcel Dekker; Jan. 7, 1991) the entire contents of which are incorporated herein by reference. 
     Protein Constituent 
     The electrochemical sensors of the invention optionally include a protein constituent disposed between the analyte sensing constituent and the analyte modulating constituent (see, e.g. element  116  in  FIG. 2 ). The term “protein constituent” is used herein according to art accepted terminology and refers to constituent containing a carrier protein or the like that is selected for compatibility with the analyte sensing constituent and/or the analyte modulating constituent. In typical embodiments, the protein constituent comprises an albumin such as human serum albumin. The HSA concentration may vary between about 0.5%-30% (w/v). Typically the HSA concentration is about 1-10% w/v, and most typically is about 5% w/v. In alternative embodiments of the invention, collagen or BSA or other structural proteins used in these contexts can be used instead of or in addition to HSA. This constituent is typically crosslinked on the analyte sensing constituent according to art accepted protocols. 
     Adhesion Promoting Constituent 
     The electrochemical sensors of the invention can include one or more adhesion promoting (AP) constituents (see, e.g. element  114  in  FIG. 2 ). The term “adhesion promoting constituent” is used herein according to art accepted terminology and refers to a constituent that includes materials selected for their ability to promote adhesion between adjoining constituents in the sensor. Typically, the adhesion promoting constituent is disposed between the analyte sensing constituent and the analyte modulating constituent. Typically, the adhesion promoting constituent is disposed between the optional protein constituent and the analyte modulating constituent. The adhesion promoter constituent can be made from any one of a wide variety of materials known in the art to facilitate the bonding between such constituents and can be applied by any one of a wide variety of methods known in the art. Typically, the adhesion promoter constituent comprises a silane compound such as γ-aminopropyltrimethoxysilane. 
     Analyte Modulating Constituent 
     The electrochemical sensors of the invention include an analyte modulating constituent disposed on the sensor (see, e.g. element  112  in  FIG. 2 ). The term “analyte modulating constituent” is used herein according to art accepted terminology and refers to a constituent that typically forms a membrane on the sensor that operates to modulate the diffusion of one or more analytes, such as glucose, through the constituent. In certain embodiments of the invention, the analyte modulating constituent is an analyte-limiting membrane which operates to prevent or restrict the diffusion of one or more analytes, such as glucose, through the constituents. In other embodiments of the invention, the analyte-modulating constituent operates to facilitate the diffusion of one or more analytes, through the constituents. Optionally such analyte modulating constituents can be formed to prevent or restrict the diffusion of one type of molecule through the constituent (e.g. glucose), while at the same time allowing or even facilitating the diffusion of other types of molecules through the constituent (e.g. O 2 ). 
     With respect to glucose sensors, in known enzyme electrodes, glucose and oxygen from blood, as well as some interferants, such as ascorbic acid and uric acid, diffuse through a primary membrane of the sensor. As the glucose, oxygen and interferants reach the analyte sensing constituent, an enzyme, such as glucose oxidase, catalyzes the conversion of glucose to hydrogen peroxide and gluconolactone. The hydrogen peroxide may diffuse back through the analyte modulating constituent, or it may diffuse to an electrode where it can be reacted to form oxygen and a proton to produce a current that is proportional to the glucose concentration. The analyte modulating sensor membrane assembly serves several functions, including selectively allowing the passage of glucose therethrough (see, e.g. U.S. Patent Application No. 2011-0152654). 
     Cover Constituent 
     The electrochemical sensors of the invention include one or more cover constituents which are typically electrically insulating protective constituents (see, e.g. element  106  in  FIG. 2 ). Typically, such cover constituents can be in the form of a coating, sheath or tube and are disposed on at least a portion of the analyte modulating constituent. Acceptable polymer coatings for use as the insulating protective cover constituent can include, but are not limited to, non-toxic biocompatible polymers such as silicone compounds, polyimides, biocompatible solder masks, epoxy acrylate copolymers, or the like. Further, these coatings can be photo-imageable to facilitate photolithographic forming of apertures through to the conductive constituent. A typical cover constituent comprises spun on silicone. As is known in the art, this constituent can be a commercially available RTV (room temperature vulcanized) silicone composition. A typical chemistry in this context is polydimethyl siloxane (acetoxy based). 
     C. Typical Analyte Sensor System Embodiments of the Invention 
     Embodiments of the sensor elements and sensors can be operatively coupled to a variety of other system elements typically used with analyte sensors (e.g. structural elements such as piercing members, insertion sets and the like as well as electronic components such as processors, monitors, medication infusion pumps and the like), for example to adapt them for use in various contexts (e.g. implantation within a mammal). One embodiment of the invention includes a method of monitoring a physiological characteristic of a user using an embodiment of the invention that includes an input element capable of receiving a signal from a sensor that is based on a sensed physiological characteristic value of the user, and a processor for analyzing the received signal. In typical embodiments of the invention, the processor determines a dynamic behavior of the physiological characteristic value and provides an observable indicator based upon the dynamic behavior of the physiological characteristic value so determined. In some embodiments, the physiological characteristic value is a measure of the concentration of blood glucose in the user. In other embodiments, the process of analyzing the received signal and determining a dynamic behavior includes repeatedly measuring the physiological characteristic value to obtain a series of physiological characteristic values in order to, for example, incorporate comparative redundancies into a sensor apparatus in a manner designed to provide confirmatory information on sensor function, analyte concentration measurements, the presence of interferences and the like. 
       FIG. 11  shows a schematic of a potentiostat that may be used to measure current in embodiments of the present invention. As shown in  FIG. 11 , a potentiostat  300  may include an op amp  310  that is connected in an electrical circuit so as to have two inputs: Vset and Vmeasured. As shown, Vmeasured is the measured value of the voltage between a reference electrode and a working electrode. Vset, on the other hand, is the optimally desired voltage across the working and reference electrodes. The current between the counter and reference electrode is measured, creating a current measurement (isig) that is output from the potentiostat. 
     Embodiments of the invention include devices which process display data from measurements of a sensed physiological characteristic (e.g. blood glucose concentrations) in a manner and format tailored to allow a user of the device to easily monitor and, if necessary, modulate the physiological status of that characteristic (e.g. modulation of blood glucose concentrations via insulin administration). An illustrative embodiment of the invention is a device comprising a sensor input capable of receiving a signal from a sensor, the signal being based on a sensed physiological characteristic value of a user; a memory for storing a plurality of measurements of the sensed physiological characteristic value of the user from the received signal from the sensor; and a display for presenting a text and/or graphical representation of the plurality of measurements of the sensed physiological characteristic value (e.g. text, a line graph or the like, a bar graph or the like, a grid pattern or the like or a combination thereof). Typically, the graphical representation displays real time measurements of the sensed physiological characteristic value. Such devices can be used in a variety of contexts, for example in combination with other medical apparatuses. In some embodiments of the invention, the device is used in combination with at least one other medical device (e.g. a glucose sensor). 
     An illustrative system embodiment consists of a glucose sensor, a transmitter and pump receiver and a glucose meter. In this system, radio signals from the transmitter can be sent to the pump receiver every 5 minutes to provide real-time sensor glucose (SG) values. Values/graphs are displayed on a monitor of the pump receiver so that a user can self monitor blood glucose and deliver insulin using their own insulin pump. Typically an embodiment of device disclosed herein communicates with a second medical device via a wired or wireless connection. Wireless communication can include for example the reception of emitted radiation signals as occurs with the transmission of signals via RF telemetry, infrared transmissions, optical transmission, sonic and ultrasonic transmissions and the like. Optionally, the device is an integral part of a medication infusion pump (e.g. an insulin pump). Typically in such devices, the physiological characteristic values include a plurality of measurements of blood glucose. 
       FIG. 3  provides a perspective view of one generalized embodiment of subcutaneous sensor insertion system and a block diagram of a sensor electronics device according to one illustrative embodiment of the invention. Additional elements typically 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.  FIG. 3  provides a perspective view of a telemetered characteristic monitor system  1 , including a subcutaneous sensor set  10  provided for subcutaneous placement of an active portion of a flexible sensor  12 , or the like, at a selected site in the body of a user. The subcutaneous or percutaneous portion of the sensor set  10  includes a hollow, slotted insertion needle  14  having a sharpened 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&#39;s bodily fluids through a window  22  formed in the cannula  16 . The sensing portion  18  is joined to a connection portion  24  that terminates in conductive contact pads, or the like, which are also exposed through one of the insulative layers. The connection portion  24  and the contact pads are generally adapted for a direct wired electrical connection to a suitable monitor  200  coupled to a display  214  for monitoring a user&#39;s condition in response to signals derived from the sensor electrodes  20 . The connection portion  24  may be conveniently connected electrically to the monitor  200  or a characteristic monitor transmitter  100  by a connector block  28  (or the like). 
     As shown in  FIG. 3 , in accordance with embodiments of the present invention, subcutaneous sensor set  10  may be configured or formed to work with either a wired or a wireless characteristic monitor system. The proximal part of the sensor  12  is mounted in a mounting base  30  adapted for placement onto the skin of a user. The mounting base  30  can be a pad having an underside surface coated with a suitable pressure sensitive adhesive layer  32 , with a peel-off paper strip  34  normally provided to cover and protect the adhesive layer  32 , until the sensor set  10  is ready for use. The mounting base  30  includes upper and lower layers  36  and  38 , with the connection portion  24  of the flexible sensor  12  being sandwiched between the layers  36  and  38 . The connection portion  24  has a forward section joined to the active sensing portion  18  of the sensor  12 , which is folded angularly to extend downwardly through a bore  40  formed in the lower base layer  38 . Optionally, the adhesive layer  32  (or another portion of the apparatus in contact with in vivo tissue) includes an anti-inflammatory agent to reduce an inflammatory response and/or anti-bacterial agent to reduce the chance of infection. The insertion needle  14  is adapted for slide-fit reception through a needle port  42  formed in the upper base layer  36  and through the lower bore  40  in the lower base layer  38 . After insertion, the insertion needle  14  is withdrawn to leave the cannula  16  with the sensing portion  18  and the sensor electrodes  20  in place at the selected insertion site. In this embodiment, the telemetered characteristic monitor transmitter  100  is coupled to a sensor set  10  by a cable  102  through a connector  104  that is electrically coupled to the connector block  28  of the connector portion  24  of the sensor set  10 . 
     In the embodiment shown in  FIG. 3 , the telemetered characteristic monitor  100  includes a housing  106  that supports a printed circuit board  108 , batteries  110 , antenna  112 , and the cable  102  with the connector  104 . In some embodiments, the housing  106  is formed from an upper case  114  and a lower case  116  that are sealed with an ultrasonic weld to form a waterproof (or resistant) seal to permit cleaning by immersion (or swabbing) with water, cleaners, alcohol or the like. In some embodiments, the upper and lower case  114  and  116  are formed from a medical grade plastic. However, in alternative embodiments, the upper case  114  and lower case  116  may be connected together by other methods, such as snap fits, sealing rings, RTV (silicone sealant) and bonded together, or the like, or formed from other materials, such as metal, composites, ceramics, or the like. In other embodiments, the separate case can be eliminated and the assembly is simply potted in epoxy or other moldable materials that is compatible with the electronics and reasonably moisture resistant. As shown, the lower case  116  may have an underside surface coated with a suitable pressure sensitive adhesive layer  118 , with a peel-off paper strip  120  normally provided to cover and protect the adhesive layer  118 , until the sensor set telemetered characteristic monitor transmitter  100  is ready for use. 
     In the illustrative embodiment shown in  FIG. 3 , the subcutaneous sensor set  10  facilitates accurate placement of a flexible thin film electrochemical sensor  12  of the type used for monitoring specific blood parameters representative of a user&#39;s condition. The sensor  12  monitors glucose levels in the body, and may be used in conjunction with automated or semi-automated medication infusion pumps of the external or implantable type as described in U.S. Pat. Nos. 4,562,751; 4,678,408; 4,685,903 or 4,573,994, to control delivery of insulin to a diabetic patient. 
     In the illustrative embodiment shown in  FIG. 3 , the sensor electrodes  10  may be used in a variety of sensing applications and may be configured in a variety of ways. For example, the sensor electrodes  10  may be used in physiological parameter sensing applications in which some type of biomolecule is used as a catalytic agent. For example, the sensor electrodes  10  may be used in a glucose and oxygen sensor having a glucose oxidase enzyme catalyzing a reaction with the sensor electrodes  20 . The sensor electrodes  10 , along with a biomolecule or some other catalytic agent, may be placed in a human body in a vascular or non-vascular environment. For example, the sensor electrodes  20  and biomolecule may be placed in a vein and be subjected to a blood stream, or may be placed in a subcutaneous or peritoneal region of the human body. 
     In the embodiment of the invention shown in  FIG. 3 , the monitor of sensor signals  200  may also be referred to as a sensor electronics device  200 . The monitor  200  may include a power source, a sensor interface, processing electronics (i.e. a processor), and data formatting electronics. The monitor  200  may be coupled to the sensor set  10  by a cable  102  through a connector that is electrically coupled to the connector block  28  of the connection portion  24 . In an alternative embodiment, the cable may be omitted. In this embodiment of the invention, the monitor  200  may include an appropriate connector for direct connection to the connection portion  104  of the sensor set  10 . The sensor set  10  may be modified to have the connector portion  104  positioned at a different location, e.g., on top of the sensor set to facilitate placement of the monitor  200  over the sensor set. 
     While the analyte sensor and sensor systems disclosed herein are typically designed to be implantable within the body of a mammal, the inventions disclosed herein are not limited to any particular environment and can instead be used in a wide variety of contexts, for example for the analysis of most in vivo and in vitro liquid samples including biological fluids such as interstitial fluids, whole-blood, lymph, plasma, serum, saliva, urine, stool, perspiration, mucus, tears, cerebrospinal fluid, nasal secretion, cervical or vaginal secretion, semen, pleural fluid, amniotic fluid, peritoneal fluid, middle ear fluid, joint fluid, gastric aspirate or the like. In addition, solid or desiccated samples may be dissolved in an appropriate solvent to provide a liquid mixture suitable for analysis. 
     It is to be understood that this invention is not limited to the particular embodiments described, as such may, of course, vary. It is also to be understood that the terminology used herein is for the purpose of describing particular embodiments only, and is not intended to be limiting, since the scope of the present invention will be limited only by the appended claims. In the description of the preferred embodiment, reference is made to the accompanying drawings which form a part hereof, and in which is shown by way of illustration a specific embodiment in which the invention may be practiced. It is to be understood that other embodiments may be utilized and structural changes may be made without departing from the scope of the present invention.