Patent Publication Number: US-2022233116-A1

Title: Systems, devices, and methods related to ketone sensors

Description:
CROSS-REFERENCE TO RELATED APPLICATIONS 
     This application claims the benefit of and priority to U.S. Provisional Patent Application Ser. No. 63/141,890, filed Jan. 26, 2021, all of which are incorporated by reference herein in their entireties and for all purposes. 
    
    
     FIELD 
     The subject matter described herein relates generally to systems, devices, and methods for determining or utilizing calibration information specific to individual medical devices such as physiological sensors, and/or the manufacturing of physiological sensors. 
     BACKGROUND 
     A vast and growing market exists for monitoring the health and condition of humans and other living animals. Information that describes the physical or physiological condition of the human can be used in countless ways to assist and improve quality of life and diagnose and treat undesirable human conditions. 
     A common device used to collect such information is a physiological sensor such as a biochemical sensor, or a device capable of sensing a chemical attribute of a biological entity. Biochemical sensors come in many forms and can be used to sense attributes in fluids, tissues, or gases forming part of or produced by a biological entity, such as a human being. These biochemical sensors can be used on or within the body itself, or they can be used on biological substances that have already been removed from the body. 
     The performance of a biochemical sensor can be characterized in a number of ways, and a characteristic of particular importance can be the accuracy of a biochemical sensor, or the degree to which the biochemical sensor correctly measures the concentration or content of the chemical being measured. The precision of the biochemical sensor, or the degree to which the measured value is exact or refined, can also be important. 
     Although biochemical sensors often have a complex and well-studied design, they can still be subject to a degree of performance variation. This can be caused by a number of factors, including variations in the manufacturing process and variations in the constituent materials used to fabricate the sensors. These variations can cause sensors of the same design and manufacturing process to have measurable differences in their performance. For these and other reasons, needs exist to improve the performance of manufactured biochemical sensors. 
     Furthermore, when cells do not receive sufficient glucose for energy production, the body begins to burn fat to generate alternate energy source, ketone bodies (ketones). The production of ketones as a source of energy can be both physiologic, such as in the case of fasting or low-carbohydrate diet, or can be detrimental such as in the case of diabetic ketoacidosis. In individuals who are on low carbohydrate (ketogenic) diets, where the carbohydrate intake is drastically reduced and replaced with fat, the body uses ketones instead of glucose for energy. A significant reduction in carbohydrates can put the body in a metabolic state called ketosis. Ketogenic diets have been used for a variety of reasons in medicine, including the management of pediatric epilepsy as well as weight loss. In patients with type 2 diabetes, nutritional ketosis is associated with sustained improvement in the atherogenic lipid and lipoprotein profile. 
     Similarly, when the body has insufficient insulin, the resulting intracellular shortage of glucose forces the body to produce ketones for fuel. However, if the ketones build up in blood, faster than they can be metabolized, the body becomes acidic. While ketoacidosis can occur in type-2 diabetic patients, it remains a significant risk in those people living with type-1 diabetes. For example, in people with diabetes managed with insulin pumps, about 3% of those between 13 and 49 years experienced more than 1 episode of diabetic ketoacidosis in the previous 3 months. 
     Currently, measurement of ketone levels is most frequently performed using urine or blood ketone test strips. However, urine or blood ketone levels using a strip-based technology has its limitations as it only provides episodic information that confirms an already ongoing ketosis or DKA event. Early identification of production of ketones may warn of impending ketoacidosis that could reduce the complications of DKA and perhaps even prevent it. Real-time continuous ketone monitoring could also help clinicians manage ketoacidosis. For individuals who are on low carbohydrate diets, the sensor may serve as a tool to monitor the effectiveness of their diet and indicate the effect of diet or exercise on the ketone levels. For these and other reasons, needs exist to improve the measurement of ketone levels. 
     SUMMARY 
     A number of example embodiments are provided herein that can be used to improve the performance of medical devices such as biochemical sensors, as well as the devices and systems utilizing these sensors. These example embodiments relate to improved techniques for assessing and predicting the performance of biochemical sensors when put to use by patients, healthcare professionals (HCPs), or other users. Many of these example embodiments pertain to the determination of calibration information based on parameters measured, recorded, or otherwise obtained during the manufacturing process. These parameters can be individualized, or specific to a discrete sensor, and the calibration information determined therefrom can likewise be individualized, or specific to that discrete sensor. 
     In many example embodiments, the calibration information is determined by also taking reference to actual tests of the sensing capability or characteristics of certain sensors. The data resulting from those tests can be used with the one or more parameters obtained during the manufacturing process to determine, estimate, extrapolate, or otherwise predict the performance of the sensor once distributed to the user. The tests, e.g., in vitro tests, used to assess sensing characteristics are often destructive, contaminatory, or otherwise of a nature that render the tested sensor unsuitable for distribution to the user. In a number of embodiments, the tests are performed on one or more sensors and the results obtained therefrom are used with the manufacturing parameter of a different, untested sensor to predict the performance of that untested sensor. In this way, the performance of a particular sensor can be predicted without subjecting the sensor to an in vitro test. 
     The information that represents the predicted performance of the sensor can be embodied as calibration information, and this calibration information can be made available to any device that seeks to use the sensing signal or data produced by the biochemical sensor to determine the end result of the measurement, e.g., the concentration or content of the substance being sensed. While applicable to smaller scales, the embodiments described herein are particularly useful when applied to high-volume manufacturing processes. For example, the embodiments described herein can be applied to groups or batches of sensors that are manufactured together. For example, in certain embodiments a subset of one or more sensors from that group or batch are subjected to in vitro testing, and the resulting test data is used with one or more manufacturing parameters obtained from a different subset of sensors of the same group or batch to predict the performance of that different subset of sensors when distributed to users. Other example embodiments are also described that incorporate one or more of the aspects described here, as well as other example embodiments that differ from that described here. 
     Also provided herein are a number of example embodiments of systems, devices, and methods for modifying a surface of a sensor substrate to aid in placement and/or sizing of a sensor element. In some of these embodiments, an area of a surface of a sensor substrate can be modified with electromagnetic radiation to create a modified area. The modified area can have a surface characteristic that is changed such that the mobility of a liquid applied to the substrate surface is either increased or decreased by the modified area. Application of a liquid to the surface of the sensor substrate can be performed such that the liquid comes to rest in a target area on the surface, where the target area is determined at least in part by the location of the modified area. The electromagnetic radiation can take various forms, such as laser radiation. In these and other embodiments, the surface modification can be the creation of a well in which a sensing element can be placed. The well can be created in various ways, such as by application of a mechanical force. Example embodiments of sensors manufactured with modified areas and/or wells are within the scope of this disclosure, as are devices, systems, and kits incorporating the same. 
     To achieve these and other advantages and in accordance with the purpose of the disclosed subject matter, as embodied and broadly described, the disclosed subject matter is directed to a system having an in vivo ketone sensor having a distal portion configured for placement in contact with an interstitial fluid of a user and a proximal portion. The sensor can include a working electrode, a sensing layer having β-hydroxybutyrate dehydrogenase, and a membrane layer configured to limit transport of one or more biomolecules. The in vivo ketone sensor can be further configured to generate signals at the working electrode corresponding to an amount of ketone in the interstitial fluid. The sensor can further include a sensor control unit having at least one contact in electrical communication with the proximal portion of the sensor and a transmitter configured to communicate with a remote device. As embodied herein, the sensor control unit can be configured to receive the generated signals, and convert the generated signals to ketone concentration data using a sensitivity associated with the in vivo ketone sensor. As embodied herein, the transmitter can be configured to communicate the ketone concentration data to the remote device. 
     As embodied herein, the membrane layer can configured to prevent the penetration of one or more interferents into a region around the working electrode. 
     As embodied herein, the remote device can include a display unit configured to display a graph of the in vivo ketone concentration over a period of time. 
     As embodied herein, the in vivo ketone sensor can be operatively coupled to the sensor control unit after the sensor is placed in contact with the interstitial fluids embodied herein, the in vivo ketone sensor can be operatively coupled to the sensor control unit before sensor placement in contact with the interstitial fluid. 
     As embodied herein, the in vivo ketone sensor can be operatively coupled to the sensor control unit before sensor placement in the interstitial fluid. In some embodiments, the sensor control unit can further include an adhesive patch having an opening through which the sensor is disposed. 
     As embodied herein, the β-hydroxybutyrate dehydrogenase can be configured to catalyze a reaction of β-hydroxybutyrate to form acetoacetate. 
     As embodied herein, the in vivo ketone sensor can further include a reference electrode including silver/silver chloride. 
     As embodied herein, the sensor control unit can be reusable. 
     Other systems, devices, methods, features, and advantages of the subject matter described herein will be or will become apparent to one with skill in the art upon examination of the following figures and detailed description. It is intended that all such additional systems, methods, features, and advantages be included within this description, be within the scope of the subject matter described herein, and be protected by the accompanying claims. In no way should the features of the example embodiments be construed as limiting the appended claims, absent express recitation of those features in the claims. 
    
    
     
       BRIEF DESCRIPTION OF FIGURES 
       The details of the subject matter set forth herein, both as to its structure and operation, may be apparent by study of the accompanying figures, in which like reference numerals refer to like parts. The components in the figures are not necessarily to scale, emphasis instead being placed upon illustrating the principles of the subject matter. Moreover, all illustrations are intended to convey concepts, where relative sizes, shapes and other detailed attributes may be illustrated schematically rather than literally or precisely. 
         FIG. 1  is a block diagram depicting an example embodiment of an in vivo analyte monitoring system. 
         FIG. 2  is a block diagram depicting an example embodiment of a data processing unit. 
         FIG. 3  is a block diagram depicting an example embodiment of a display device. 
         FIG. 4  as a schematic diagram depicting an example embodiment of an analyte sensor. 
         FIG. 5A  is a perspective view depicting an example embodiment of an analyte sensor penetrating through the skin. 
         FIG. 5B  is a cross sectional view depicting a portion of the analyte sensor of  FIG. 5A . 
         FIGS. 6-9  are cross-sectional views depicting example embodiments of analyte sensors. 
         FIG. 10A  is a cross-sectional view depicting an example embodiment of an analyte sensor. 
         FIGS. 10B-10C  are cross-sectional views depicting example embodiments of analyte sensors as viewed from line A-A of  FIG. 10A . 
         FIG. 11  is a conceptual view depicting an example embodiment of an analyte monitoring system. 
         FIG. 12  is a block diagram depicting an example embodiment of on body electronics. 
         FIG. 13  is a block diagram depicting an example embodiment of a display device. 
         FIG. 14  is a flow diagram depicting an example embodiment of information exchange within and analyte monitoring system. 
         FIG. 15  is a graph depicting an example of an in vitro sensitivity of an analyte sensor. 
         FIG. 16  shows the signal output for a D-3-hydroxybutyrate dehydrogenase sensor over the course of 2.3 hours at varying concentrations of D-3-hydroxybutyrate according to certain embodiments. 
         FIG. 17  depicts the linearity of the sensor signal of a D-3-hydroxybutyrate dehydrogenase sensor as a function of D-3-hydroxybutyrate concentration. 
         FIG. 18  shows the signal output for a D-3-hydroxybutyrate dehydrogenase sensor using free NAD over the course of 3.6 hours at varying concentrations of D-3-hydroxybutyrate according to certain embodiments. 
         FIG. 19  depicts the linearity of the sensor signal of a D-3-hydroxybutyrate dehydrogenase sensor as a function of D-3-hydroxybutyrate concentration (Ketone). 
         FIG. 20  depicts the stability of the sensor signal of a D-3-hydroxybutyrate dehydrogenase sensor. 
         FIG. 21  depicts the stability of the sensor signal of a free NAD and immobilized NAD sensor. 
         FIG. 22  is an example plot of change in sensor response with sequential addition of ketone aliquots. 
         FIG. 23  is an example plot of calibrated sensor response as a function of ketone concentration. 
         FIG. 24  is an example plot of change in sensor response. 
         FIG. 25  is an example plot of response of three ketone sensors simultaneously worn by one subject with changing concentration of ketone in the body. 
         FIG. 26A-26G  is an example plot of ketone values in interstitial fluid measured by example sensors against capillary ketone strip reference measurements. 
     
    
    
     DETAILED DESCRIPTION 
     The present subject matter is described in detail with reference to example embodiments. These example embodiments are set forth for illustrative purposes to aid those of ordinary skill in the art in understanding and appreciating the full scope of the present subject matter. These example embodiments do not constitute an exhaustive recitation of all manners in which the present subject matter can be implemented, as such an exhaustive recitation is both burdensome and unnecessary in light of the example embodiments explicitly set forth. As such, the present subject matter is of a breadth that extends beyond those particular embodiments explicitly set forth herein. 
     The subject matter described herein generally relates to advancements in techniques for calibrating medical devices capable of sensing one or more biochemical attributes, as well as systems and devices for performing these calibration techniques. In many embodiments, the techniques permit the determination of individualized calibration information that varies between and is particular to individual medical devices, as opposed to a single calibration value that is determined for groups of medical devices as a whole. There are many classes of medical devices that sense biochemical attributes, and thus there are many applications with which this subject matter can be utilized. Several of these classes of medical devices will be described herein, but these are merely examples and do not constitute an exhaustive recitation of all classes of medical devices with which the present subject matter finds utility. 
     Medical devices capable of sensing or monitoring chemical levels in bodily fluids can often be classified as part of either in vivo systems or in vitro systems. In vivo systems include one or more medical devices that sense one or more biochemical attributes of bodily fluid that is within the human body, often by partially or wholly implanting the medical device (e.g., a sensor) within the human body. A common example is an in vivo analyte sensor useful in monitoring analyte levels in the human body. These analyte sensors can be designed to detect glucose or other analytes that are particularly relevant in monitoring a diabetic condition. 
     In vitro systems include one or more medical devices that sense one or more biochemical attributes of bodily fluid, such as blood, plasma, urine, etc., that has been removed from the human body, or other substances such as a homogenized biopsy sample. In vitro systems can also be referred to as ex vivo systems. A common example is an in vitro analyte sensor such as a test strip. In vitro test strips can also be designed to detect and measure glucose or other analytes that are particularly relevant for monitoring a diabetic condition. 
     Systems and devices incorporating or utilizing data from either in vivo or in vitro medical devices are broadly referred to herein as biochemical monitoring systems and biochemical monitoring devices, respectively. Systems and devices incorporating or utilizing data from medical devices that are designed to sense the level of an analyte (e.g., glucose) are referred to herein as analyte monitoring systems and analyte monitoring devices, respectively. 
     Example embodiments relating to these calibration techniques will be presented by reference to their application to in vivo medical devices and in vitro medical devices. The majority of the embodiments are described with respect to in vivo medical devices, particularly, in vivo analyte sensors. This is merely to facilitate the presentation of the features and aspects of these example embodiments, and is not intended to limit these calibration techniques to use with only in vivo analyte sensors. Indeed, as noted already, the present subject matter is broadly applicable to other types of medical devices, a number of embodiments of which will also be explicitly described. 
     Certain example embodiments relating to these calibration techniques permit the determination of individualized calibration information specific to an individual sensor and, if desired, the subsequent use of that individualized calibration information to calibrate an output of the individual sensor. In many embodiments, the individualized calibration information is specific to each individual medical device within a common manufacturing group or lot and can vary between each individual medical device with the common group. These embodiments are in contrast to approaches where a single calibration value is determined for a group or lot of medical devices as a whole such that every medical device in the common manufacturing group has the same calibration value. 
     In some example embodiments, a sensing characteristic of a first subset (e.g., a sample or baseline subset) of medical devices is determined. For analyte sensors, this sensing characteristic can be, e.g., a sensitivity of the sensor to the analyte. The sensing characteristic can be determined with in vitro (or in vivo use) testing of the first subset of medical devices. Examples of such testing will be described in more detail herein. One or more individualized manufacturing parameter can be measured from each medical device in a different second subset of medical devices (e.g., a distribution subset intended for distribution from the manufacturer to third party users). In some example embodiments, the baseline and distribution subsets are taken from the same production lot. The measurement of the individualized manufacturing parameter can be performed by, e.g., the manufacturer during or after the manufacturing process. The individualized manufacturing parameter can directly or indirectly correlate to the sensing characteristic of the medical device, and numerous examples of such individualized manufacturing parameters are described herein. 
     Individualized calibration information can then be independently determined for each medical device within the distribution subset of medical devices using at least the individualized manufacturing parameter of each device within the distribution subset and the sensing characteristic of the baseline subset. This can result in calibration information that is specific to each medical device in the distribution subset and that can vary between the medical devices from variation of the individualized manufacturing parameter. In some embodiments, two or more individualized manufacturing parameters are used to determine the calibration information. In some embodiments, one or more qualitative manufacturing parameters are used, either alone or in conjunction with a quantitative individualized manufacturing parameter. 
     As will be discussed in further detail herein, studies have confirmed that embodiments of the present subject matter result in tangible improvements in the accuracy of biochemical sensing measurements made by the medical devices. This represents an improvement in the operation of the calibrated medical devices themselves, which in turn results in an improvement in the operation of the monitoring systems and/or monitoring devices incorporating these medical devices, as well as an improvement in the operation of the computing devices that process or otherwise utilize the improved accuracy data produced by the calibrated medical devices. Improvements through lessening variations between medical devices were also confirmed, as were improvements to the manufacturing yield of the medical devices. 
     Before describing the embodiments relating to individualized calibration techniques in detail, it is first desirable to describe example embodiments of in vivo analyte monitoring systems and in vitro analyte monitoring systems, as well as examples of their operations, all of which can be used with embodiments of these calibration techniques. 
     Example Embodiments of In Vivo Analyte Monitoring Systems 
     There are various types of analyte monitoring systems used with in vivo sensors. “Continuous Analyte Monitoring” systems (e.g., “Continuous Glucose Monitoring” systems), for example, are in vivo systems that can transmit data from a sensor control device to a reader device repeatedly or continuously without prompting, e.g., automatically according to a schedule. “Flash Analyte Monitoring” systems (e.g., “Flash Glucose Monitoring” systems or simply “Flash” systems), as another example, are in vivo systems that can transfer data from a sensor control device in response to a scan or request for data by a reader device, such as with a Near Field Communication (NFC) or Radio Frequency Identification (RFID) protocol. 
     An in vivo analyte sensor can be partially or wholly implanted within the human body such that it makes contact with the bodily fluid in the user and senses the analyte levels therein. The in vivo sensor can be part of a sensor control device that resides on the body of the user and contains the electronics and power supply that enable and control the analyte sensing. The sensor control device, and variations thereof, can also be referred to as a “sensor control unit,” an “on-body electronics” device or unit, an “on-body” device or unit, a “sensor data communication” device or unit, or a transmitter device or unit, to name a few. The term “on body” or “on-body” refers to any device that resides directly on the body or in close proximity to the body, such as a wearable device (e.g., glasses, armband, wristband or bracelet, neckband, or necklace, etc.). 
     In vivo monitoring systems can also include one or more reader devices that receive sensed analyte data from the sensor control device. These reader devices can process, retransmit, and/or display the sensed analyte data, in any number of forms. These devices, and variations thereof, can be referred to as “handheld reader devices,” “reader devices” (or simply, “readers”), “display devices,” “handheld electronics” (or handhelds), “portable data processing” devices or units, “data receivers,” “receiver” devices or units (or simply receivers), “relay” devices or units, “remote” devices or units, “companion” devices or units, “human interface” devices or units, to name a few. Computing devices such as personal computers can be used as a reader device. 
     In vivo analyte monitoring systems can be used with in vitro medical devices as well. For example, a reader device can incorporate or be coupled with a port for receiving an in vitro test strip carrying a bodily fluid of the user, which can be analyzed to determine the user&#39;s analyte level. 
     In Vivo Sensors 
     In vivo sensors can be formed on a substrate, e.g., a substantially planar substrate, or a non-planar rounded or cylindrical substrate. In many embodiments, the sensor comprises at least one electrically conductive structure, e.g., an electrode. Sensor embodiments can be single electrode embodiments (e.g., having no more than one electrode), or multiple electrode embodiments (e.g., having exactly two, exactly three, or more electrodes). Embodiments of the sensor will often include a working electrode, and can also include at least one counter electrode (or counter/reference electrode), and/or at least one reference electrode (or at reference/counter electrode). Electrodes can be arranged as discrete regions electrically isolated by insulative regions, and can be electrically connected to circuitry for receiving (and optionally conditioning and/or processing) the electrical signals produced by the electrodes. Electrodes can have planar (e.g., relatively flat) surfaces or non-planar (e.g., relatively curved or rounded, such as semi-hemispherical, cylindrical, or irregular surfaces and combinations thereof). Electrodes can be arranged in layers or concentrically or otherwise. 
     Accordingly, embodiments include analyte monitoring devices and systems that include an analyte sensor at least a portion of which is positionable beneath the skin surface of the user for the in vivo detection of an analyte, including glucose, lactate, and the like, in a body fluid. Embodiments include wholly implantable analyte sensors and analyte sensors in which only a portion of the sensor is positioned under the skin and a portion of the sensor resides above the skin, e.g., for contact to a sensor control device (which may include a transmitter), a receiver/display unit, transceiver, processor, etc. The sensor may be, for example, positionable through an exterior skin surface of a user for the continuous or periodic monitoring (periodic according to a regular interval, an irregular interval, a schedule, frequent repeats, etc.) of a level of an analyte in the user&#39;s bodily fluid (e.g., interstitial fluid, subcutaneous fluid, dermal fluid, blood, or other bodily fluid of interest). For the purposes of this description, continuous monitoring and periodic monitoring will be used interchangeably, unless noted otherwise. The sensor response may be correlated and/or converted to analyte levels in blood or other fluids. In certain embodiments, an analyte sensor may be positioned in contact with interstitial fluid to detect the level of glucose, which detected glucose may be used to infer the glucose level in the user&#39;s bloodstream. Analyte sensors may be insertable into a vein, artery, or other portion of the body containing fluid. Embodiments of the analyte sensors may be configured for monitoring the level of the analyte over a time period which may range from seconds, minutes, hours, days, weeks, to months, or longer. 
     In certain embodiments, the analyte sensors, such as glucose sensors, are capable of in vivo detection of an analyte for one hour or more, e.g., a few hours or more, e.g., a few days or more, e.g., three or more days, e.g., five days or more, e.g., seven days or more, e.g., several weeks or more, or one month or more. Future analyte levels may be predicted based on information obtained, e.g., the current analyte level at time t0, the rate of change of the analyte, etc. Predictive alarms may notify the user of predicted analyte levels that may be of concern in advance of the user&#39;s analyte level reaching the future predicted analyte level. This provides the user an opportunity to take corrective action. 
     In an electrochemical embodiment, the sensor is placed, transcutaneously, for example, into a subcutaneous site such that subcutaneous fluid of the site comes into contact with the sensor. In other in vivo embodiments, placement of at least a portion of the sensor may be in a blood vessel. The sensor operates to electrolyze an analyte of interest in the subcutaneous fluid or blood such that a current is generated between the working electrode and the counter electrode. A value for the current associated with the working electrode is determined. If multiple working electrodes are used, current values from each of the working electrodes may be determined. A microprocessor may be used to collect these periodically determined current values or to further process these values. 
     If an analyte concentration is successfully determined, it may be displayed, stored, transmitted, and/or otherwise processed to provide useful information. By way of example, raw signal or analyte concentrations may be used as a basis for determining a rate of change in analyte concentration, which should not change at a rate greater than a predetermined threshold amount. If the rate of change of analyte concentration exceeds the predefined threshold, an indication maybe displayed or otherwise transmitted to indicate this fact. In certain embodiments, an alarm is activated to alert a user if the rate of change of analyte concentration exceeds the predefined threshold. 
     As demonstrated herein, the present embodiments are useful in connection with a device that is used to measure or monitor an analyte (e.g., glucose), such as any such device described herein. The embodiments described herein can be used to monitor and/or process information regarding any number of one or more different analytes. Analytes that may be monitored include, but are not limited to, acetyl choline, amylase, bilirubin, carbon dioxide, cholesterol, chorionic gonadotropin, glycosylated hemoglobin (HbA1c), creatine kinase (e.g., CK-MB), creatine, creatinine, DNA, fructosamine, glucose, glucose derivatives, glutamine, growth hormones, hormones, ketones, ketone bodies, lactate, oxygen, peroxide, prostate-specific antigen, proteins, prothrombin, RNA, thyroid stimulating hormone, troponin, and any combination thereof. The concentration of drugs, such as, for example, antibiotics (e.g., gentamicin, vancomycin, and the like), digitoxin, digoxin, drugs of abuse, theophylline, and warfarin, may be monitored in addition to or instead of analytes. In embodiments that monitor more than one analyte, the analytes may be monitored at the same or different times. These methods may also be used in connection with a device that is used to measure or monitor another analyte (e.g., ketones, ketone bodies, HbA1c, and the like), including oxygen, carbon dioxide, proteins, drugs, or another moiety of interest, for example, or any combination thereof, found in bodily fluid, including subcutaneous fluid, dermal fluid, interstitial fluid, or other bodily fluid of interest, for example, or any combination thereof. In general, the device is in good contact, such as thorough and substantially continuous contact, with the bodily fluid. 
     According to embodiments, the analyte sensor may be operatively coupled to the sensor control device/unit after sensor placement in contact with interstitial fluid. In some embodiments, the analyte sensor may be operatively coupled to the sensor control device/unit before sensor placement in contact with interstitial fluid. 
     According to embodiments of the present disclosure, the measurement sensor is one suited for electrochemical measurement of analyte concentration, for example ketone concentration, in a bodily fluid. In these embodiments, the measurement sensor includes at least a working electrode and a counter electrode. Other embodiments may further include a reference electrode. The working electrode is typically associated with a β-hydroxybutyrate (BHB)-responsive enzyme. A mediator may also be included. In some embodiments, a mediator is added to the sensor by a manufacturer, e.g., is included with the sensor prior to use. The redox mediator may be disposed relative to the working electrode and is capable of transferring electrons between a compound and a working electrode, either directly or indirectly. The redox mediator may be, for example, immobilized on the working electrode, e.g., entrapped on a surface or chemically bound to a surface. 
     Embodiments of the subject disclosure include in vivo analyte monitoring devices, systems, kits, and processes of analyte monitoring and making analyte monitoring devices, systems, and kits. Included are on-body (e.g., at least a portion of a device, system or a component thereof is maintained on the body of or in close proximity to a user to monitor an analyte), physiological monitoring devices configured for real time measurement/monitoring of desired analyte level such as a glucose level over one or more predetermined time periods such as one or more predetermined monitoring time periods. Embodiments include transcutaneously positioned analyte sensors that are electrically coupled with electronics provided in a housing that is designed to be attached to the body of a user, for example, to a skin surface of a user, during the usage life of the analyte sensors or predetermined monitoring time periods. For example, on body electronics assembly include electronics that are operatively coupled to an analyte sensor and provided in a housing for placement on the body of a user. 
     Such device and system with analyte sensors provide continuous or periodic analyte level monitoring that is executed automatically, or semi-automatically by control logic or routines programmed or programmable in the monitoring devices or systems. As used herein, continuous, automatic, and/or periodic monitoring refer to the in vivo monitoring or detection of analyte levels with transcutaneously positioned analyte sensors. 
     In certain embodiments, the results of the in vivo monitored analyte level are automatically communicated from an electronics unit to another device or component of the system. That is, when the results are available, the results are automatically transmitted to a display device (or other user interaction device) of the system, for example, according to a fixed or dynamic data communication schedule executed by the system. In other embodiments, the results of the in vivo monitored analyte level are not automatically communicated, transferred, or output to one or more device or component of the system. In such embodiments, the results are provided only in response to a query to the system. That is, the results are communicated to a component or a device of the system only in response to the query or request for such results. In certain embodiments, the results of the in vivo monitoring may be logged or stored in a memory of the system and only communicated or transferred to another device or component of the system after the one or more predetermined monitoring time periods. 
     Embodiments include software and/or hardware to transform any one of the devices, components, or systems into any one of the other devices, components, or systems, where such transformation may be user-configurable after manufacture. Transformation modules that include hardware and/or software to accomplish such transformation may be mateable to a given system to transform it. 
     Embodiments include electronics coupled to analyte sensors that provide functionalities to operate the analyte sensors for monitoring analyte levels over a predetermined monitoring time period such as for example, about 30 days (or more in certain embodiments), about 14 days, about 10, about 5 days, about 1 day, less than about 1 day. In certain embodiments, the usage life of each analyte sensor may be the same as or different from the predetermined monitoring time periods. Components of the electronics to provide the functionalities to operate the analyte sensors in certain embodiments include control logic or microprocessors coupled to a power supply such as a battery to drive the in vivo analyte sensors to perform electrochemical reactions to generate resulting signals that correspond to the monitored analyte levels. 
     Electronics may also include other components such as one or more data storage units or memory (volatile and/or non-volatile), communication component(s) to communicate information corresponding to the in vivo monitored analyte level to a display device automatically when the information is available, or selectively in response to a request for the monitored analyte level information. Data communication between display devices and the electronics coupled to the sensor in certain embodiments are implemented serially (e.g., data transfer between them are not performed at the same time), or in parallel. For example, the display device in certain embodiments is configured to transmit a signal or data packet to the electronics coupled to the sensor, and upon receipt of the transmitted signal or data packet, the electronics coupled to the sensor communicates back to the display device. In certain embodiments, a display device may be configured to provide RF power and data/signals continually, and detecting or receiving one or more return data packet or signal from electronics coupled to the sensor when it is within a predetermined RF power range from the display device. In certain embodiments, the display device and the electronics coupled to the sensor may be configured to transmit one or more data packets at the same time. 
     Embodiments also include electronics programmed to store or log in the one or more data storage units or a memory data associated with the monitored analyte level over the sensor usage life or during a monitoring time period. During the monitoring time period, information corresponding to the monitored analyte level may be stored but not displayed or output during the sensor usage life, and the stored data may be later retrieved from memory at the end of the sensor usage life or after the expiration of the predetermined monitoring time period, e.g., for clinical analysis, therapy management, etc. 
     In certain embodiments, the predetermined monitoring time period is the same as the sensor usage life time period such that when an analyte sensor usage life expires (thus no longer used for in vivo analyte level monitoring), the predetermined monitoring time period ends. In certain embodiments, the predetermined monitoring time period may include multiple sensor usage life time periods such that when an analyte sensor usage life expires, the predetermined monitoring time period has not ended, and the expired analyte sensor is replaced with another analyte sensor during the same predetermined monitoring time period. The predetermined monitoring time period in certain embodiments includes the replacement of multiple analyte sensors for use. 
     Analyte level trend information in certain embodiments is generated or constructed based on stored analyte level information spanning a time period (e.g., corresponding to a temperature time period, or other) and communicated to the display device. The trend information in certain embodiments is output graphically and/or audibly and/or tactilely, and/or numerically and/or otherwise presented on a user interface of the display device to provide indication of the analyte level variation during this time period. 
     Embodiments include wirelessly communicating analyte level information from an on body electronics device to a second device such as a display device. Examples of communication protocols between on body electronics and the display device may include radio frequency identification (RFID) protocols or RF communication protocols. Example RFID protocols include but are not limited to Near Field Communication (NFC) protocols that include short communication ranges (e.g., about 12 inches or less, or about 6 inches or less, or about 3 inches or less, or about 2 inches or less), high frequency wireless communication protocols, far field communication protocols (e.g., using ultra high frequency (UHF) communication systems) for providing signals or data from on body electronics to display devices. 
     Communication protocols in certain embodiments use 433 MHz frequency, 13.56 MHz frequency, 2.45 GHz frequency, or other suitable frequencies for wireless communication between the on body electronics that includes electronics coupled to an analyte sensor, and one or more display devices and/or other devices such as a personal computer. While certain data transmission frequencies and/or data communication ranges are described above, within the scope of the present disclosure, other data suitable data transmission frequencies and/or data communication ranges can be used between the various devices in the analyte monitoring system. 
     Embodiments include data management systems including, for example, a data network and/or personal computer and/or a server terminal and/or one or more remote computers that are configured to receive collected or stored data from the display device for presenting analyte information and/or further processing in conjunction with the physiological monitoring for health management. For example, a display device may include one or more communication ports (hard wired or wireless) for connection to a data network or a computer terminal to transfer collected or stored analyte related data to another device and/or location. Analyte related data in certain embodiments are directly communicated from the electronics coupled to the analyte sensor to a personal computer, server terminal, and/or remote computers over the data network. 
     In certain embodiments, analyte information is only provided or evident to a user (provided at a user interface device) when desired by the user even though an in vivo analyte sensor automatically and/or continuously monitors the analyte level in vivo, e.g., the sensor automatically monitors analyte such as ketone on a pre-defined time interval over its usage life. For example, an analyte sensor may be positioned in vivo and coupled to on body electronics for a given sensing period, e.g., about 14 days, about 21 days, or about 30 days or more. In certain embodiments, the sensor-derived analyte information is automatically communicated from the sensor electronics assembly to a remote monitor device or display device for output to a user throughout the 14 day period according to a schedule programmed at the on body electronics (e.g., about every 1 minute or about every 5 minutes or about every 10 minutes, or the like). In certain embodiments, sensor-derived analyte information is only communicated from the sensor electronics assembly to a remote monitor device or display device at user-determined times, e.g., whenever a user decides to check analyte information. At such times, a communications system is activated and sensor-derived information is then sent from the on body electronics to the remote device or display device. For example, using RFID communication, in one embodiment, the user positions the display device in close proximity to the on body electronics coupled to the analyte sensor and receives the real time (and/or historical) analyte level information from the on body electronics (herein after referred to as “on demand” reading). 
     In still other embodiments, the information may be communicated from a first device to a second device automatically and/or continuously when the analyte information is available, and the second device stores or logs the received information without presenting or outputting the information to the user. In such embodiments, the information is received by the second device from the first device when the information becomes available (e.g., when the sensor detects the analyte level according to a time schedule). However, the received information is initially stored in the second device and only output to a user interface or an output component of the second device (e.g., display) upon detection of a request for the information on the second device. 
     Accordingly, in certain embodiments once a sensor electronics assembly is placed on the body so that at least a portion of the in vivo sensor is in contact with bodily fluid and the sensor is electrically coupled to the electronics unit, sensor derived analyte information may be communicated from the on body electronics to a display device on-demand by powering on the display device (or it may be continually powered), and executing a software algorithm stored in and accessed from a memory of the display device, to generate one or more request commands, control signal or data packet to send to the on body electronics. The software algorithm executed under, for example, the control of the microprocessor or application specific integrated circuit (ASIC) of the display device may include routines to detect the position of the on body electronics relative to the display device to initiate the transmission of the generated request command, control signal and/or data packet. 
     Display devices may also include programming stored in memory for execution by one or more microprocessors and/or ASICs to generate and transmit the one or more request command, control signal or data packet to send to the on body electronics in response to a user activation of an input mechanism on the display device such as depressing a button on the display device, triggering a soft button associated with the data communication function, and so on. The input mechanism may be alternatively or additionally provided on or in the on body electronics which may be configured for user activation. In certain embodiments, voice commands or audible signals may be used to prompt or instruct the microprocessor or ASIC to execute the software routine(s) stored in the memory to generate and transmit the one or more request command, control signal or data packet to the on body device. In the embodiments that are voice activated or responsive to voice commands or audible signals, on body electronics and/or display device includes a microphone, a speaker, and processing routines stored in the respective memories of the on body electronics and/or the display device to process the voice commands and/or audible signals. In certain embodiments, positioning the on body device and the display device within a predetermined distance (e.g., close proximity) relative to each other initiates one or more software routines stored in the memory of the display device to generate and transmit a request command, control signal or data packet. 
     Different types and/or forms and/or amounts of information may be sent for each on demand reading, including but not limited to one or more of current analyte level information (e.g., real time or the most recently obtained analyte level information temporally corresponding to the time the reading is initiated), rate of change of an analyte over a predetermined time period, rate of the rate of change of an analyte (acceleration in the rate of change), historical analyte information corresponding to analyte information obtained prior to a given reading and stored in memory of the assembly. Some or all of real time, historical, rate of change, rate of rate of change (such as acceleration or deceleration) information may be sent to a display device for a given reading. In certain embodiments, the type and/or form and/or amount of information sent to a display device may be preprogrammed and/or unchangeable (e.g., preset at manufacturing), or may not be preprogrammed and/or unchangeable so that it may be selectable and/or changeable in the field one or more times (e.g., by activating a switch of the system, etc.). 
     Accordingly, in certain embodiments, for each on demand reading, a display device will output a current (real time) sensor-derived analyte value (e.g., in numerical format), a current rate of analyte change (e.g., in the form of an analyte rate indicator such as an arrow pointing in a direction to indicate the current rate), and analyte trend history data based on sensor readings acquired by and stored in memory of on body electronics (e.g., in the form of a graphical trace). Additionally, the on skin or sensor temperature reading or measurement associated with each on demand reading may be communicated from the on body electronics to the display device. The temperature reading or measurement, however, may not be output or displayed on the display device, but rather, used in conjunction with a software routine executed by the display device to correct or compensate the analyte measurement output to the user on the display device. 
     As described, embodiments include in vivo analyte sensors and on body electronics that together provide body wearable sensor electronics assemblies. In certain embodiments, in vivo analyte sensors are fully integrated with on body electronics (fixedly connected during manufacture), while in other embodiments they are separate but connectable post manufacture (e.g., before, during or after sensor insertion into a body). On body electronics may include an in vivo ketone sensor, electronics, battery, and antenna encased (except for the sensor portion that is for in vivo positioning) in a waterproof housing that includes or is attachable to an adhesive pad. In certain embodiments, the housing withstands immersion in about one meter of water for up to at least 30 minutes. In certain embodiments, the housing withstands continuous underwater contact, e.g., for longer than about 30 minutes, and continues to function properly according to its intended use, e.g., without water damage to the housing electronics where the housing is suitable for water submersion. 
     Embodiments include sensor insertion devices, which also may be referred to herein as sensor delivery units, or the like. Insertion devices may retain on body electronics assemblies completely in an interior compartment, e.g., an insertion device may be “pre-loaded” with on body electronics assemblies during the manufacturing process (e.g., on body electronics may be packaged in a sterile interior compartment of an insertion device). In such embodiments, insertion devices may form sensor assembly packages (including sterile packages) for pre-use or new on body electronics assemblies, and insertion devices configured to apply on body electronics assemblies to recipient bodies. 
     Embodiments include portable handheld display devices, as separate devices and spaced apart from an on body electronics assembly, that collect information from the assemblies and provide sensor derived analyte readings to users. Such devices can be referred to in a number of ways that have already been set forth. Certain embodiments may include an integrated in vitro analyte meter. In certain embodiments, display devices include one or more wired or wireless communications ports such as USB, serial, parallel, or the like, configured to establish communication between a display device and another unit (e.g., on body electronics, power unit to recharge a battery, a PC, etc.). For example, a display device communication port may enable charging a display device battery with a respective charging cable and/or data exchange between a display device and its compatible informatics software. 
     Compatible informatics software in certain embodiments include, for example, but not limited to stand alone or network connection enabled data management software program, resident or running on a display device, personal computer, a server terminal, for example, to perform data analysis, charting, data storage, data archiving and data communication as well as data synchronization. Informatics software in certain embodiments may also include software for executing field upgradable functions to upgrade firmware of a display device and/or on body electronics unit to upgrade the resident software on the display device and/or the on body electronics unit, e.g., with versions of firmware that include additional features and/or include software bugs or errors fixed, etc. 
     Embodiments include programming embedded on a computer readable medium, e.g., computer-based application software (may also be referred to herein as informatics software or programming or the like) that processes analyte information obtained from the system and/or user self-reported data. Application software may be installed on a host computer such as a mobile telephone, PC, an Internet-enabled human interface device such as an Internet-enabled phone, personal digital assistant, or the like, by a display device or an on body electronics unit. Informatics programming may transform data acquired and stored on a display device or on body unit for use by a user. 
     As described in detail below, embodiments include devices, systems, kits and/or methods to monitor one or more physiological parameters such as, for example, but not limited to, analyte levels, temperature levels, heart rate, user activity level, over a predetermined monitoring time period. Also provided are methods of manufacturing. Predetermined monitoring time periods may be less than about 1 hour, or may include about 1 hour or more, e.g., about a few hours or more, e.g., about a few days of more, e.g., about 3 or more days, e.g., about 5 days or more, e.g., about 7 days or more, e.g., about 10 days or more, e.g., about 14 days or more, e.g., about several weeks, e.g., about 1 month or more. In certain embodiments, after the expiration of the predetermined monitoring time period, one or more features of the system may be automatically deactivated or disabled at the on body electronics assembly and/or display device. 
     For example, a predetermined monitoring time period may begin with positioning the sensor in vivo and in contact with a bodily fluid such as interstitial fluid, and/or with the initiation (or powering on to full operational mode) of the on body electronics. Initialization of on body electronics may be implemented with a command generated and transmitted by a display device in response to the activation of a switch and/or by placing the display device within a predetermined distance (e.g., close proximity) to the on body electronics, or by user manual activation of a switch on the on body electronics unit, e.g., depressing a button, or such activation may be caused by the insertion device, e.g., as described in U.S. Patent Publication No. 2011/0213225A1, the disclosure of which is incorporated by reference in its entirety. 
     When initialized in response to a received command from a display device, the on body electronics retrieves and executes from its memory software routine to fully power on the components of the on body electronics, effectively placing the on body electronics in full operational mode in response to receiving the activation command from the display device. For example, prior to the receipt of the command from the display device, a portion of the components in the on body electronics may be powered by its internal power supply such as a battery while another portion of the components in the on body electronics may be in powered down or low power including no power, inactive mode, or all components may be in an inactive mode, powered down mode. Upon receipt of the command, the remaining portion (or all) of the components of the on body electronics is switched to active, fully operational mode. 
     Embodiments of on body electronics may include one or more printed circuit boards with electronics including control logic implemented in ASIC, microprocessors, memory, and the like, and transcutaneously positionable analyte sensors forming a single assembly. On body electronics may be configured to provide one or more signals or data packets associated with a monitored analyte level upon detection of a display device of the analyte monitoring system within a predetermined proximity for a period of time (for example, about 2 minutes, e.g., 1 minute or less, e.g., about 30 seconds or less, e.g., about 10 seconds or less, e.g., about 5 seconds or less, e.g., about 2 seconds or less) and/or until a confirmation, such as an audible and/or visual and/or tactile (e.g., vibratory) notification, is output on the display device indicating successful acquisition of the analyte related signal from the on body electronics. A distinguishing notification may also be output for unsuccessful acquisition in certain embodiments. 
     In certain embodiments, the monitored analyte level may be correlated and/or converted to ketone levels in blood or other bodily fluids. Such conversion may be accomplished by the on body electronics, but in other embodiments, will be accomplished with display device electronics. 
     Referring now to  FIG. 1 , the analyte monitoring system  100  includes an analyte sensor  101 , a data processing unit  102  connectable to the sensor  101 , and a primary receiver unit or display device  104 . In some instances, the primary display device  104  is configured to communicate with the data processing unit  102  via a communication link  103 . In certain embodiments, the primary display device  104  may be further configured to transmit data to a data processing terminal  105  to evaluate or otherwise process or format data received by the primary display device  104 . The data processing terminal  105  may be configured to receive data directly from the data processing unit  102  via a communication link  107 , which may optionally be configured for bi-directional communication. Further, the data processing unit  102  may include electronics and a transmitter or a transceiver to transmit and/or receive data to and/or from the primary display device  104  and/or the data processing terminal  105  and/or optionally a secondary receiver unit or display device  106 . 
     Also shown in  FIG. 1  is an optional secondary display device  106  which is operatively coupled to the communication link  103  and configured to receive data transmitted from the data processing unit  102 . The secondary display device  106  may be configured to communicate with the primary display device  104 , as well as the data processing terminal  105 . In certain embodiments, the secondary display device  106  may be configured for bi-directional wireless communication with each of the primary display device  104  and the data processing terminal  105 . As discussed in further detail below, in some instances, the secondary display device  106  may be a de-featured receiver as compared to the primary display device  104 , for instance, the secondary display device  106  may include a limited or minimal number of functions and features as compared with the primary display device  104 . As such, the secondary display device  106  may include a smaller (in one or more, including all, dimensions), compact housing or embodied in a device including a wrist watch, arm band, PDA, mp3 player, cell phone, etc., for example. Alternatively, the secondary display device  106  may be configured with the same or substantially similar functions and features as the primary display device  104 . The secondary display device  106  may include a docking portion configured to mate with a docking cradle unit for placement by, e.g., the bedside for night time monitoring, and/or a bi-directional communication device. A docking cradle may recharge a power supply. 
     Only one analyte sensor  101 , data processing unit  102  and data processing terminal  105  are shown in the embodiment of the analyte monitoring system  100  illustrated in  FIG. 1 . However, it will be appreciated by one of ordinary skill in the art that the analyte monitoring system  100  may include more than one sensor  101  and/or more than one data processing unit  102 , and/or more than one data processing terminal  105 . Multiple sensors may be positioned in a user for analyte monitoring at the same or different times. In certain embodiments, analyte information obtained by a first sensor positioned in a user may be employed as a comparison to analyte information obtained by a second sensor. This may be useful to confirm or validate analyte information obtained from one or both of the sensors. Such redundancy may be useful if analyte information is contemplated in critical therapy-related decisions. In certain embodiments, a first sensor may be used to calibrate a second sensor. 
     In a multi-component environment, each component may be configured to be uniquely identified by one or more of the other components in the system so that communication conflict may be readily resolved between the various components within the analyte monitoring system  100 . For example, unique IDs, communication channels, and the like, may be used. 
     In certain embodiments, the sensor  101  is physically positioned in or on the body of a user whose analyte level is being monitored. The sensor  101  may be configured to at least periodically sample the analyte level of the user and convert the sampled analyte level into a corresponding signal for transmission by the data processing unit  102 . The data processing unit  102  is coupleable to the sensor  101  so that both devices are positioned in or on the user&#39;s body, with at least a portion of the analyte sensor  101  positioned transcutaneously. The data processing unit  102  may include a fixation element, such as an adhesive or the like, to secure it to the user&#39;s body. A mount (not shown) attachable to the user and mateable with the data processing unit  102  may be used. For example, a mount may include an adhesive surface. The data processing unit  102  performs data processing functions, where such functions may include, but are not limited to, filtering and encoding of data signals, each of which corresponds to a sampled analyte level of the user, for transmission to the primary display device  104  via the communication link  103 . In some embodiments, the sensor  101  or the data processing unit  102  or a combined sensor/data processing unit may be wholly implantable under the skin surface of the user. 
     In certain embodiments, the primary display device  104  may include an analog interface section including an RF receiver and an antenna that is configured to communicate with the data processing unit  102  via the communication link  103 , and a data processing section for processing the received data from the data processing unit  102  including data decoding, error detection and correction, data clock generation, data bit recovery, etc., or any combination thereof. 
     In operation, the primary display device  104  in certain embodiments is configured to synchronize with the data processing unit  102  to uniquely identify the data processing unit  102 , based on, for example, an identification information of the data processing unit  102 , and thereafter, to periodically receive signals transmitted from the data processing unit  102  associated with the monitored analyte levels monitored by the sensor  101 . 
     Referring again to  FIG. 1 , the data processing terminal  105  may include a personal computer, a portable computer including a laptop or a handheld device (e.g., a personal digital assistant (PDA), a telephone including a cellular phone (e.g., a multimedia and Internet-enabled mobile phone including an iPhone®, a Blackberry®, an Android phone, or similar phone), an mp3 player (e.g., an iPOD™, etc.), a pager, and the like), and/or a drug delivery device (e.g., an infusion device), each of which may be configured for data communication with the display devices via a wired or a wireless connection. Additionally, the data processing terminal  105  may further be connected to a data network (not shown) for storing, retrieving, updating, and/or analyzing data corresponding to the detected analyte level of the user. 
     The data processing terminal  105  may include a drug delivery device (e.g., an infusion device) such as an insulin infusion pump or the like, which may be configured to administer a drug (e.g., insulin) to the user, and which may be configured to communicate with the primary display device  104  for receiving, among others, the measured analyte level. Alternatively, the primary display device  104  may be configured to integrate an infusion device therein so that the primary display device  104  is configured to administer an appropriate drug (e.g., insulin) to users, for example, for administering and modifying basal profiles, as well as for determining appropriate boluses for administration based on, among others, the detected analyte levels received from the data processing unit  102 . An infusion device may be an external device or an internal device, such as a device wholly implantable in a user. 
     In certain embodiments, the data processing terminal  105 , which may include an infusion device, e.g., an insulin pump, may be configured to receive the analyte signals from the data processing unit  102 , and thus, incorporate the functions of the primary display device  104  including data processing for managing the user&#39;s insulin therapy and analyte monitoring. In certain embodiments, the communication link  103 , as well as one or more of the other communication interfaces shown in  FIG. 1 , may use one or more wireless communication protocols, such as, but not limited to: an RF communication protocol, an infrared communication protocol, a Bluetooth enabled communication protocol, an 802.11x wireless communication protocol, or an equivalent wireless communication protocol which would allow secure, wireless communication of several units (for example, per Health Insurance Portability and Accountability Act (HIPPA) requirements), while avoiding potential data collision and interference. 
       FIG. 2  is a block diagram depicting an embodiment of a data processing unit  102  of the analyte monitoring system shown in  FIG. 1 . User input and/or interface components may be included or a data processing unit may be free of user input and/or interface components. In certain embodiments, one or more application-specific integrated circuits (ASIC) (e.g., having processing circuitry and non-transitory memory for storing software instructions for execution by the processing circuitry) may be used to implement one or more functions or routines associated with the operations of the data processing unit (and/or display device) using for example one or more state machines and buffers. 
     As can be seen in the embodiment of  FIG. 2 , the analyte sensor  101  ( FIG. 1 ) includes four contacts, three of which are electrodes: a working electrode (W)  210 , a reference electrode (R)  212 , and a counter electrode (C)  213 , each operatively coupled to the analog interface  201  of the data processing unit  102 . This embodiment also shows an optional guard contact (G)  211 . Fewer or greater electrodes may be employed. For example, the counter and reference electrode functions may be served by a single counter/reference electrode. In some cases, there may be more than one working electrode and/or reference electrode and/or counter electrode, etc. 
       FIG. 3  is a block diagram of an embodiment of a receiver/monitor unit such as the primary display device  104  of the analyte monitoring system shown in  FIG. 1 . The primary display device  104  includes one or more of: a test strip interface  301 , an RF receiver  302 , a user input  303 , an optional temperature detection section  304 , and a clock  305 , each of which is operatively coupled to a processing and storage section  307  (that can include processing circuitry and non-transitory memory storing software instructions for execution by the processing circuitry). The primary display device  104  also includes a power supply  306  operatively coupled to a power conversion and monitoring section  308 . Further, the power conversion and monitoring section  308  is also coupled to the processing and storage section  307 . Moreover, also shown are a receiver serial communication section  309 , and an output  310 , each operatively coupled to the processing and storage section  307 . The primary display device  104  may include user input and/or interface components or may be free of user input and/or interface components. 
     In certain embodiments, the test strip interface  301  includes an analyte testing portion (e.g., a ketone level testing portion) to receive a blood (or other body fluid sample) analyte test or information related thereto. For example, the test strip interface  301  may include a test strip port to receive a test strip (e.g., a ketone test strip). The device may determine the analyte level of the test strip, and optionally display (or otherwise notice) the analyte level on the output  310  of the primary display device  104 . Any suitable test strip may be employed, e.g., test strips that only require a very small amount (e.g., 3 microliters or less, e.g., 1 microliter or less, e.g., 0.5 microliters or less, e.g., 0.1 microliters or less), of applied sample to the strip in order to obtain accurate glucose information. Ketone information obtained by an in vitro glucose testing device may be used for a variety of purposes, computations, etc. For example, the information may be used to calibrate sensor  101  ( FIG. 1 ), confirm results of sensor  101  to increase the confidence thereof (e.g., in instances in which information obtained by sensor  101  is employed in therapy related decisions), etc. 
     In further embodiments, the data processing unit  102  and/or the primary display device  104  and/or the secondary display device  106 , and/or the data processing terminal/infusion device  105  may be configured to receive the analyte value wirelessly over a communication link from, for example, a blood glucose meter. In further embodiments, a user manipulating or using the analyte monitoring system  100  may manually input the analyte value using, for example, a user interface (for example, a keyboard, keypad, voice commands, and the like) incorporated in one or more of the data processing unit  102 , the primary display device  104 , secondary display device  106 , or the data processing terminal/infusion device  105 . 
       FIG. 4  schematically shows an embodiment of an analyte sensor  400  in accordance with the embodiments of the present disclosure. This sensor embodiment includes electrodes  401 ,  402  and  403  on a base  404 . Electrodes (and/or other features) may be applied or otherwise processed using any suitable technology, e.g., chemical vapor deposition (CVD), physical vapor deposition, sputtering, reactive sputtering, printing, coating, ablating (e.g., laser ablation), painting, dip coating, etching, and the like. Materials include, but are not limited to, any one or more of aluminum, carbon (including graphite), cobalt, copper, gallium, gold, indium, iridium, iron, lead, magnesium, mercury (as an amalgam), nickel, niobium, osmium, palladium, platinum, rhenium, rhodium, selenium, silicon (e.g., doped polycrystalline silicon), silver, tantalum, tin, titanium, tungsten, uranium, vanadium, zinc, zirconium, mixtures thereof, and alloys, oxides, or metallic compounds of these elements. 
     The analyte sensor  400  may be wholly implantable in a user or may be configured so that only a portion is positioned within (internal) a user and another portion outside (external) a user. For example, the sensor  400  may include a first portion positionable above a surface of the skin  410 , and a second portion positioned below the surface of the skin. In such embodiments, the external portion may include contacts (connected to respective electrodes of the second portion by traces) to connect to another device also external to the user such as a sensor control device. While the embodiment of  FIG. 4  shows three electrodes side-by-side on the same surface of base  404 , other configurations are contemplated, e.g., fewer or greater electrodes, some or all electrodes on different surfaces of the base or present on another base, some or all electrodes stacked together, electrodes of differing materials and dimensions, etc. 
       FIG. 5A  shows a perspective view of an embodiment of an analyte sensor  500  having a first portion (which in this embodiment may be characterized as a major portion) positionable above a surface of the skin  510 , and a second portion (which in this embodiment may be characterized as a minor portion) that includes an insertion tip  530  positionable below the surface of the skin, e.g., penetrating through the skin and into, e.g., the subcutaneous space  520 , in contact with the user&#39;s biofluid, such as interstitial fluid. Contact portions of a working electrode  511 , a reference electrode  512 , and a counter electrode  513  are positioned on the first portion of the sensor  500  situated above the skin surface  510 . A working electrode  501 , a reference electrode  502 , and a counter electrode  503  are shown at the second portion of the sensor  500  and particularly at the insertion tip  530 . Traces may be provided from the electrodes at the tip  530  to the contacts, as shown in  FIG. 5A . It is to be understood that greater or fewer electrodes may be provided on a sensor. For example, a sensor may include more than one working electrode and/or the counter and reference electrodes may be a single counter/reference electrode, etc. 
       FIG. 5B  shows a cross sectional view of a portion of the sensor  500  of  FIG. 5A . The electrodes  501 ,  509 / 502  and  503 , of the sensor  500  as well as the substrate and the dielectric layers are provided in a layered configuration or construction. For example, as shown in  FIG. 5B , in one embodiment, the sensor  500  (such as the analyte sensor  101  of  FIG. 1 ), includes a substrate layer  504 , and a first conducting layer  501  such as carbon, gold, etc., disposed on at least a portion of the substrate layer  504 , and which may provide the working electrode. Also shown disposed on at least a portion of the first conducting layer  501  is a sensing region  508 . 
     A first insulation layer  505 , such as a first dielectric layer in certain embodiments, is disposed or layered on at least a portion of the first conducting layer  501 , and further, a second conducting layer  509  may be disposed or stacked on top of at least a portion of the first insulation layer (or dielectric layer)  505 . As shown in  FIG. 5B , the second conducting layer  509  in conjunction with a second conducting material  502 , such as a layer of silver/silver chloride (Ag/AgCl), may provide the reference electrode. 
     A second insulation layer  506 , such as a second dielectric layer in certain embodiments, may be disposed or layered on at least a portion of the second conducting layer  509 . Further, a third conducting layer  503  may be disposed on at least a portion of the second insulation layer  506  and may provide the counter electrode  503 . Finally, a third insulation layer  507  may be disposed or layered on at least a portion of the third conducting layer  503 . In this manner, the sensor  500  may be layered such that at least a portion of each of the conducting layers is separated by a respective insulation layer (for example, a dielectric layer). The embodiments of  FIGS. 5A and 5B  show the layers having different lengths. In certain instances, some or all of the layers may have the same or different lengths and/or widths. 
     In certain embodiments, some or all of the electrodes  501 ,  502 ,  503  may be provided on the same side of the substrate  504  in the layered construction as described above, or alternatively, may be provided in a co-planar manner such that two or more electrodes may be positioned on the same plane (e.g., side-by side (e.g., parallel) or angled relative to each other) on the substrate  504 . For example, co-planar electrodes may include a suitable spacing therebetween and/or include a dielectric material or insulation material disposed between the conducting layers/electrodes. 
     Furthermore, in certain embodiments, one or more of the electrodes  501 ,  502 ,  503  may be disposed on opposing sides of the substrate  504 . In such embodiments, contact pads may be on the same or different sides of the substrate. For example, an electrode may be on a first side and its respective contact may be on a second side, e.g., a trace connecting the electrode and the contact may traverse through the substrate. 
     Embodiments of a double-sided, stacked sensor configuration which may be utilized in connection with the present disclosure are described below with reference to  FIGS. 6-8 .  FIG. 6  shows a cross-sectional view of a distal portion of a double-sided analyte sensor  600 . Analyte sensor  600  includes an at least generally planar insulative base substrate  601 , e.g., an at least generally planar dielectric base substrate, having a first conductive layer  602  which substantially covers the entirety of a first surface area, e.g., the top surface area, of insulative substrate  601 , e.g., the conductive layer substantially extends the entire length of the substrate to the distal edge and across the entire width of the substrate from side edge to side edge. A second conductive layer  603  substantially covers the entirety of a second surface, e.g., the bottom side, of insulative base substrate  601 . However, one or both of the conductive layers may terminate proximally of the distal edge and/or may have a width which is less than that of insulative substrate  601  where the width ends a selected distance from the side edges of the substrate, which distance may be equidistant or vary from each of the side edges. 
     One of the first or second conductive layers, e.g., first conductive layer  602 , may be configured to include the sensor&#39;s working electrode. The opposing conductive layer, here, second conductive layer  603 , may be configured to include a reference and/or counter electrode. Where conductive layer  603  serves as either a reference or counter electrode, but not both, a third electrode may optionally be provided either on a surface area of the proximal portion of the sensor (not shown), on a separate substrate, or as an additional conductive layer positioned either above or below conductive layer  602  or  603  and separated from those layers by an insulative layer or layers. For example, in some embodiments, where analyte sensor  600  is configured to be partially implanted, conductive layer  603  may be configured to include a reference electrode, and a third electrode (not shown) and present only on a non-implanted proximal portion of the sensor may be configured to include the sensor&#39;s counter electrode. 
     A first insulative layer  604  covers at least a portion of conductive layer  602  and a second insulative layer  605  covers at least a portion of conductive layer  603 . In one embodiment, at least one of first insulative layer  604  and second insulative layer  605  does not extend to the distal end of analyte sensor  600  leaving an exposed region of the conductive layer or layers. 
       FIG. 7  shows a cross-sectional view of a distal portion of a double-sided analyte sensor  700  including an at least generally planar insulative base substrate  701 , e.g., an at least generally planar dielectric base substrate, having a first conductive layer  702  which substantially covers the entirety of a first surface area, e.g., the top surface area, of insulative substrate  701 , e.g., the conductive layer substantially extends the entire length of the substrate to the distal edge and across the entire width of the substrate from side edge to side edge. A second conductive layer  703  substantially covers the entirety of a second surface, e.g., the bottom side, of insulative base substrate  701 . However, one or both of the conductive layers may terminate proximally of the distal edge and/or may have a width which is less than that of insulative substrate  701  where the width ends a selected distance from the side edges of the substrate, which distance may be equidistant or vary from each of the side edges. 
     In the embodiment of  FIG. 7 , conductive layer  702  is configured to include a working electrode which includes a sensing region  702 A disposed on at least a portion of the first conductive layer  702  as shown and as discussed in greater detail below. While a single sensing region  702 A is shown, it should be noted that in other embodiments a plurality of spatially separated sensing elements may be utilized. 
     In the embodiment of  FIG. 7 , conductive layer  703  is configured to include a reference electrode which includes a secondary layer of conductive material  703 A, e.g., Ag/AgCl, disposed over a distal portion of conductive layer  703 . 
     A first insulative layer  704  covers a portion of conductive layer  702  and a second insulative layer  705  covers a portion of conductive layer  703 . First insulative layer  704  does not extend to the distal end of analyte sensor  700  leaving an exposed region of the conductive layer where the sensing region  702 A is positioned. The insulative layer  705  on the bottom/reference electrode side of the sensor, may extend any suitable length of the sensor&#39;s distal section, e.g., it may extend the entire length of both of the primary and secondary conductive layers or portions thereof. For example, as illustrated in  FIG. 7 , bottom insulative layer  705  extends over the entire bottom surface area of secondary conductive material  703 A but terminates proximally of the distal end of the length of the conductive layer  703 . It is noted that at least the ends of the secondary conductive material  703 A which extend along the side edges of the substrate  701  are not covered by insulative layer  705  and, as such, are exposed to the environment when in operative use. 
     In an alternative embodiment, as shown in  FIG. 8 , analyte sensor  800  has an insulative layer  804  on the working electrode side of an insulative base substrate  801 , which may be provided prior to sensing region  802 A whereby the insulative layer  804  has at least two portions spaced apart from each other on conductive layer  802 . The sensing region  802 A is then provided in the spacing between the two portions. More than two spaced apart portions may be provided, e.g., where a plurality of sensing components or layers is desired. Bottom insulative layer  805  has a length which terminates proximally of secondary conductive layer  803 A on bottom primary conductive layer  803 . Additional conducting and dielectric layers may be provided on either or both sides of the sensors, as described above. 
     While  FIGS. 6-8  depict or are discussed herein as capable of providing the working and reference electrodes in a particular layered configuration, it should be noted that the relative positioning of these layers may be modified. For example, a counter electrode layer may be provided on one side of an insulative base substrate while working and reference electrode layers are provided in a stacked configuration on the opposite side of the insulative base substrate. In addition, a different number of electrodes may be provided than depicted in  FIGS. 6-8  by adjusting the number of conductive and insulative layers. For example, a 3 or four electrode sensor may be provided. 
     One or more membranes, which may function as one or more of an analyte flux modulating layer and/or an interferent-eliminating layer and/or biocompatible layer, discussed in greater detail below, may be included with, on or about the sensor, e.g., as one or more of the outermost layer(s). For example, the membrane layer can be configured to prevent penetration of one or more interferents into a region around the working electrode. Those of ordinary skill in the art will readily recognize that the membrane can take many forms. The membrane can include just one component, or multiple components. The membrane can have a globular shape, such as if encompassing a terminal region of the sensor (e.g., the lateral sides and terminal tip). The membrane can have a generally planar structure, and can be characterized as a layer. Planar membranes can be smooth or can have minor surface (topological) variations. The membrane can also be configured as other non-planar structures. For example, the membrane can have a cylindrical shape or a partially cylindrical shape, a hemispherical shape or other partially spherical shape, an irregular shape, or other rounded or curved shape. 
     In certain embodiments, as illustrated in  FIG. 7 , a first membrane layer  706  may be provided solely over the sensing region  702 A on the working electrode  702  to modulate the rate of diffusion or flux of the analyte to the sensing region. For embodiments in which a membrane layer is provided over a single component/material, it may be suitable to do so with the same striping configuration and method as used for the other materials/components. Here, the membrane material  706  preferably has a width greater than that of sensing component  702 A. As it acts to limit the flux of the analyte to the sensor&#39;s active area, and thus contributes to the sensitivity of the sensor, controlling the thickness of membrane  706  is important. Providing membrane  706  in the form of a stripe/band facilitates control of its thickness. A second membrane layer  707 , which coats the remaining surface area of the sensor tail, may also be provided to serve as a biocompatible conformal coating and provide smooth edges over the entirety of the sensor. In other sensor embodiments, as illustrated in  FIG. 8 , a single, homogenous membrane  806  may be coated over the entire sensor surface area, or at least over both sides of the distal tail portion. It is noted that to coat the distal and side edges of the sensor, the membrane material may have to be applied subsequent to singulation of the sensor precursors. In some embodiments, the analyte sensor is dip-coated following singulation to apply one or more membranes. Alternatively, the analyte sensor could be slot-die coated wherein each side of the analyte sensor is coated separately. 
       FIG. 9  shows a cross-sectional view of a distal portion of an example double-sided analyte sensor  900  according to one embodiment of the present disclosure, wherein the double-sided analyte sensor includes an at least generally planar insulative base substrate  901 , e.g., an at least generally planar dielectric base substrate, having a first conductive layer  902 . A second conductive layer  903  is positioned on a first side, e.g., the bottom side, of insulative base substrate  901 . While depicted as extending to the distal edge of the sensor, one or both of the conductive layers may terminate proximally of the distal edge and/or may have a width which is less than that of insulative substrate  901  where the width ends a selected distance from the side edges of the substrate, which distance may be equidistant or vary from each of the side edges. See, for example, the analyte sensor assembly  900 , discussed in more detail below, wherein first and second conductive layers are provided which define electrodes, including, e.g., electrode traces, which have widths which are less than that of the insulative base substrate. 
     In the embodiment of  FIG. 9 , conductive layer  903  is configured to include a working electrode which includes a sensing region  908  disposed on at least a portion of the conductive layer  903 , which sensing region is discussed in greater detail below. It should be noted that a plurality of spatially separated sensing components or layers may be utilized in forming the working electrode, e.g., one or more sensing “dots” or areas may be provided on the conductive layer  903 , as shown herein, or a single sensing component may be used (not shown). 
     In the embodiment of  FIG. 9 , conductive layer  906  is configured to include a reference electrode which includes a secondary layer of conductive material  906 A, e.g., Ag/AgCl, disposed on a distal portion of conductive layer  906 . Like conductive layers  902  and  903 , conductive layer  906  may terminate proximally of the distal edge and/or may have a width which is less than that of insulative substrate  901  where the width ends a selected distance from the side edges of the substrate, which distance may be equidistant or vary from each of the side edges, as discussed in greater detail below in reference to  FIGS. 10A-10C . 
     In the embodiment shown in  FIG. 9 , conductive layer  902  is configured to include a counter electrode. A first insulative layer  904  covers a portion of conductive layer  902  and a second insulative layer  905  covers a portion of conductive layer  903 . First insulative layer  904  does not extend to the distal end of analyte sensor  900  leaving an exposed region of the conductive layer  902  which acts as the counter electrode. An insulative layer  905  covers a portion of conductive layer  903  leaving an exposed region of the conductive layer  903  where the sensing region  908  is positioned. As discussed above, multiple spatially separated sensing components or layers may be provided (as shown) in some embodiments, while in other embodiments a single sensing region may be provided. The insulative layer  907  on a first side, e.g., the bottom side of the sensor (in the view provided by  FIG. 9 ), may extend any suitable length of the sensor&#39;s distal section, e.g., it may extend the entire length of both of conductive layers  906  and  906 A or portions thereof. For example, as illustrated in  FIG. 9 , bottom insulative layer  907  extends over the entire bottom surface area of secondary conductive material  906 A and terminates distally of the distal end of the length of the conductive layer  906 . It is noted that at least the ends of the secondary conductive material  906 A which extend along the side edges of the substrate  901  are not covered by insulative layer  907  and, as such, are exposed to the environment when in operative use. 
     As illustrated in  FIG. 9 , a homogenous membrane  909  may be coated over the entire sensor surface area, or at least over both sides of the distal tail portion. It is noted that to coat the distal and side edges of the sensor, the membrane material may have to be applied subsequent to singulation of the sensor precursors. In some embodiments, the analyte sensor is dip-coated following singulation to apply one or more membranes (or to apply one membrane in various stages). Alternatively, the analyte sensor could be slot-die coated wherein each side of the analyte sensor is coated separately. Membrane  909  is shown in  FIG. 9  as having a squared shape matching the underlying surface variations, but can have a more globular or amorphous shape as well. 
     When manufacturing layered sensors, it may be desirable to utilize relatively thin insulative layers to reduce total sensor width. For example, with reference to  FIG. 9 , insulative layers  904 ,  905  and  907  may be relatively thin relative to insulative substrate layer  901 . For example, insulative layers  904 ,  905  and  907  may have a thickness in the range of 20-25 μm while substrate layer  901  has a thickness in the range of 0.1 to 0.15 mm. However, during singulation of the sensors where such singulation is accomplished by cutting through two or more conductive layers which are separated by such thin insulative layers, shorting between the two conductive layers may occur. 
     One method of addressing this potential issue is to provide one of the conductive layers, e.g., electrodes layers, at least in part as a relatively narrow electrode, including, e.g., a relatively narrow conductive trace, such that during the singulation process the sensor is cut on either side of the narrow electrode such that one electrode is cut without cutting through the narrow electrode. 
     For example, with reference to  FIGS. 10A-10C , a sensor  1000  is depicted which includes insulative layers  1003  and  1005 . Insulative layers  1003  and  1005  may be thin relative to generally planar insulative base substrate layer  1001 , or vice versa. For example, insulative layers  1003  and  1005  may have a thickness in the range of 15-30 μm while substrate layer  1001  has a thickness in the range of 0.1 to 0.15 mm. Such sensors may be manufactured in sheets wherein a single sheet includes a plurality of sensors. However, such a process generally requires singulation of the sensors prior to use. Where such singulation requires cutting through two or more conductive layers which are separated by insulative layers, shorting between the two conductive layers may occur, particularly if the insulative layers are thin. In order to avoid such shorting, fewer than all of the conductive layers may be cut through during the singulation process. For example, at least one of the conductive layers may be provided at least in part as an electrode, e.g., including a conductive trace, having a narrow width relative to one or more other conductive layers such that during the singulation process a first conductive layer separated from a second conductive layer only by a thin insulative layer, e.g., an insulative layer having a thickness in the range of 15-30 μm, is cut while a second conductive layer is not. 
     For example, with reference to  FIGS. 10A and 10C , a sensor  1000  includes an at least generally planar insulative base substrate  1001 . Positioned on the at least generally planar insulative base substrate  1001  is a first conductive layer  1002 . A first relatively thin insulative layer  1003 , e.g., an insulative layer having a thickness in the range of 15-30 μm, is positioned on the first conductive layer  1002  and second conductive layer  1004  is positioned on the relatively thin insulative layer  1003 . Finally, a second relatively thin insulative layer  1005 , e.g., an insulative layer having a thickness in the range of 15-30 μm, is positioned on the second conductive layer  1004 . 
     As shown in  FIG. 10B , first conductive layer  1002  may be an electrode having a narrow width relative to conductive layer  1004  as shown in the  FIG. 10B  cross-section taken at lines A-A. Alternatively, second conductive layer  1004  may be a conductive electrode having a narrow width relative to conductive layer  1002  as shown in the  FIG. 1C  cross-section taken at lines A-A. Singulation cut lines  1006  are shown in  FIGS. 10B and 10C . The sensor may be singulated, for example, by cutting to either side of the relatively narrow conductive electrode, e.g., in regions  1007 , as shown in  FIGS. 10B and 10C . With reference to  FIG. 10B , singulation by cutting along singulation cut lines  1006  results in cutting through conductive layer  1004  but not conductive layer  1002 . With reference to  FIG. 10C , singulation by cutting along singulation cut lines  1006  results in cutting through conductive layer  1002  but not conductive layer  1004 . 
     An embodiment of a sensing region may be described as the area shown schematically in  FIG. 5B  as  508  and  FIG. 9  as  908 . As noted above the sensing region may be provided as a single sensing component as shown in  FIG. 5B  as  508 ,  FIG. 7  as  702 A and  FIG. 8  as  802 A, or provided as a plurality of sensing components as shown in  FIG. 9  as  908 . A plurality of sensing components or sensing “spots” is described in US Patent Application Publication No. 2012/0150005, incorporated by reference herein in its entirety. 
     The term “sensing region” is a broad term and may be described as the active chemical area of the biosensor. Those of ordinary skill in the art will readily recognize that the sensing region can take many forms. The sensing region can include just one component, or multiple components (e.g., such as sensing region  908  of  FIG. 9 ). In the embodiment of  FIG. 5B , for example, the sensing region is a generally planar structure, and can be characterized as a layer. Planar sensing regions can be smooth or can have minor surface (topological) variations. The sensing region can also be a non-planar structure. For example, the sensing region can have a cylindrical shape or a partially cylindrical shape, a hemispherical shape or other partially spherical shape, an irregular shape, or other rounded or curved shape. 
     In certain instances, the analyte-responsive enzyme is distributed throughout the sensing region. For example, the analyte-responsive enzyme may be distributed uniformly throughout the sensing region, such that the concentration of the analyte-responsive enzyme is substantially the same throughout the sensing region. In some cases, the sensing region may have a homogeneous distribution of the analyte-responsive enzyme. In certain embodiments, the redox mediator is distributed throughout the sensing region. For example, the redox mediator may be distributed uniformly throughout the sensing region, such that the concentration of the redox mediator is substantially the same throughout the sensing region. In some cases, the sensing region may have a homogeneous distribution of the redox mediator. In certain embodiments, both the analyte-responsive enzyme and the redox mediator are distributed uniformly throughout the sensing region, as described above. 
     As noted above, analyte sensors may include an analyte-responsive enzyme to provide a sensing component or sensing region. Some analytes, such as oxygen, can be directly electrooxidized or electroreduced on a sensor, and more specifically at least on a working electrode of a sensor. Other analytes, such as glucose and lactate, require the presence of at least one electron transfer agent and/or at least one catalyst to facilitate the electrooxidation or electroreduction of the analyte. Catalysts may also be used for those analytes, such as oxygen, that can be directly electrooxidized or electroreduced on the working electrode. For these analytes, each working electrode includes a sensing region (see for example sensing region  508  of  FIG. 5B ) proximate to or on a surface of a working electrode. In many embodiments, a sensing region is formed near or on only a small portion of at least a working electrode. 
     The sensing region can include one or more components constructed to facilitate the electrochemical oxidation or reduction of the analyte. The sensing region may include, for example, a catalyst to catalyze a reaction of the analyte and produce a response at the working electrode, an electron transfer agent to transfer electrons between the analyte and the working electrode (or other component), or both. 
     A variety of different sensing region configurations may be used. The sensing region is often located in contact with or in proximity to an electrode, such as the working electrode. In certain embodiments, the sensing region is deposited on the conductive material of the working electrode. The sensing region may extend beyond the conductive material of the working electrode. In some cases, the sensing region may also extend over other electrodes, e.g., over the counter electrode and/or reference electrode (or if a counter/reference is provided). 
     A sensing region that is in direct contact with the working electrode may contain an electron transfer agent to transfer electrons directly or indirectly between the analyte and the working electrode, and/or a catalyst to facilitate a reaction of the analyte. For example, a glucose, lactate, or oxygen electrode may be formed having a sensing region which contains a catalyst, including glucose oxidase, glucose dehydrogenase, lactate oxidase, or laccase, respectively, and an electron transfer agent that facilitates the electrooxidation of the glucose, lactate, or oxygen, respectively. 
     In other embodiments, the sensing region is not deposited directly on the working electrode. Instead, the sensing region  508  ( FIG. 5 ), for example, may be spaced apart from the working electrode, and separated from the working electrode, e.g., by a separation layer. A separation layer may include one or more membranes or films or a physical distance. In addition to separating the working electrode from the sensing region, the separation layer may also act as a mass transport limiting layer and/or an interferent eliminating layer and/or a biocompatible layer. 
     In certain embodiments which include more than one working electrode, one or more of the working electrodes may not have a corresponding sensing region, or may have a sensing region which does not contain one or more components (e.g., an electron transfer agent and/or catalyst) needed to electrolyze the analyte. Thus, the signal at this working electrode may correspond to background signal which may be removed from the analyte signal obtained from one or more other working electrodes that are associated with fully-functional sensing regions by, for example, subtracting the signal. 
     In certain embodiments, the sensing region includes one or more electron transfer agents. Electron transfer agents that may be employed are electroreducible and electrooxidizable ions or molecules having redox potentials that are a few hundred millivolts above or below the redox potential of the standard calomel electrode (SCE). The electron transfer agent may be organic, organometallic, or inorganic. Examples of organic redox species are quinones and species that in their oxidized state have quinoid structures, such as Nile blue and indophenol. Examples of organometallic redox species are metallocenes including ferrocene. Examples of inorganic redox species are hexacyanoferrate (III), ruthenium hexamine, etc. Additional examples include those described in U.S. Pat. Nos. 6,736,957, 7,501,053 and 7,754,093, the disclosures of each of which are incorporated herein by reference in their entirety. 
     In certain embodiments, electron transfer agents have structures or charges which prevent or substantially reduce the diffusional loss of the electron transfer agent during the period of time that the sample is being analyzed. For example, electron transfer agents include but are not limited to a redox species, e.g., bound to a polymer which can in turn be disposed on or near the working electrode. The bond between the redox species and the polymer may be covalent, coordinative, or ionic. Although any organic, organometallic or inorganic redox species may be bound to a polymer and used as an electron transfer agent, in certain embodiments the redox species is a transition metal compound or complex, e.g., osmium, ruthenium, iron, and cobalt compounds or complexes. It will be recognized that many redox species described for use with a polymeric component may also be used, without a polymeric component. 
     Embodiments of polymeric electron transfer agents may contain a redox species covalently bound in a polymeric composition. An example of this type of mediator is poly(vinylferrocene). Another type of electron transfer agent contains an ionically-bound redox species. This type of mediator may include a charged polymer coupled to an oppositely charged redox species. Examples of this type of mediator include a negatively charged polymer coupled to a positively charged redox species such as an osmium or ruthenium polypyridyl cation. Another example of an ionically-bound mediator is a positively charged polymer including quaternized poly(4-vinyl pyridine) or poly(1-vinyl imidazole) coupled to a negatively charged redox species such as ferricyanide or ferrocyanide. In other embodiments, electron transfer agents include a redox species coordinatively bound to a polymer. For example, the mediator may be formed by coordination of an osmium or cobalt 2,2′-bipyridyl complex to poly(1-vinyl imidazole) or poly(4-vinyl pyridine). 
     Suitable electron transfer agents are osmium transition metal complexes with one or more ligands, each ligand having a nitrogen-containing heterocycle such as 2,2′-bipyridine, 1,10-phenanthroline, 1-methyl, 2-pyridyl biimidazole, or derivatives thereof. The electron transfer agents may also have one or more ligands covalently bound in a polymer, each ligand having at least one nitrogen-containing heterocycle, such as pyridine, imidazole, or derivatives thereof. One example of an electron transfer agent includes (a) a polymer or copolymer having pyridine or imidazole functional groups and (b) osmium cations complexed with two ligands, each ligand containing 2,2′-bipyridine, 1,10-phenanthroline, or derivatives thereof, the two ligands not necessarily being the same. Some derivatives of 2,2′-bipyridine for complexation with the osmium cation include but are not limited to 4,4′-dimethyl-2,2′-bipyridine and mono-, di-, and polyalkoxy-2,2′-bipyridines, including 4,4′-dimethoxy-2,2′-bipyridine. Derivatives of 1,10-phenanthroline for complexation with the osmium cation include but are not limited to 4,7-dimethyl-1,10-phenanthroline and mono, di-, and polyalkoxy-1,10-phenanthrolines, such as 4,7-dimethoxy-1,10-phenanthroline. Polymers for complexation with the osmium cation include but are not limited to polymers and copolymers of poly(1-vinyl imidazole) (referred to as “PVI”) and poly(4-vinyl pyridine) (referred to as “PVP”). Suitable copolymer substituents of poly(1-vinyl imidazole) include acrylonitrile, acrylamide, and substituted or quaternized N-vinyl imidazole, e.g., electron transfer agents with osmium complexed to a polymer or copolymer of poly(1-vinyl imidazole). 
     Embodiments may employ electron transfer agents having a redox potential ranging from about −200 mV to about +200 mV versus the standard calomel electrode (SCE). The sensing region may also include a catalyst which is capable of catalyzing a reaction of the analyte. The catalyst may also, in some embodiments, act as an electron transfer agent. One example of a suitable catalyst is an enzyme which catalyzes a reaction of the analyte. For example, a catalyst, including a glucose oxidase, glucose dehydrogenase (e.g., pyrroloquinoline quinone (PQQ), dependent glucose dehydrogenase, flavine adenine dinucleotide (FAD) dependent glucose dehydrogenase, or nicotinamide adenine dinucleotide (NAD) dependent glucose dehydrogenase), may be used when the analyte of interest is glucose. A lactate oxidase or lactate dehydrogenase may be used when the analyte of interest is lactate. Laccase may be used when the analyte of interest is oxygen or when oxygen is generated or consumed in response to a reaction of the analyte. 
     In certain embodiments, a catalyst may be attached to a polymer, cross linking the catalyst with another electron transfer agent, which, as described above, may be polymeric. A second catalyst may also be used in certain embodiments. This second catalyst may be used to catalyze a reaction of a product compound resulting from the catalyzed reaction of the analyte. The second catalyst may operate with an electron transfer agent to electrolyze the product compound to generate a signal at the working electrode. Alternatively, a second catalyst may be provided in an interferent-eliminating layer to catalyze reactions that remove interferents. 
     In certain embodiments, the sensor operates at a low oxidizing potential, e.g., a potential of about +40 mV vs. Ag/AgCl. This sensing region uses, for example, an osmium (Os)-based mediator constructed for low potential operation. Accordingly, in certain embodiments the sensing element is a redox active component that includes (1) osmium-based mediator molecules that include (bidente) ligands, and (2) glucose oxidase enzyme molecules. These two constituents are combined together in the sensing region of the sensor. 
     A mass transport limiting layer (not shown), e.g., an analyte flux modulating layer, may be included with the sensor to act as a diffusion-limiting barrier to reduce the rate of mass transport of the analyte, for example, glucose or lactate or ketone, into the region around the working electrodes. The mass transport limiting layers are useful in limiting the flux of an analyte to a working electrode in an electrochemical sensor so that the sensor is linearly responsive over a large range of analyte concentrations and is easily calibrated. Mass transport limiting layers may include polymers and may be biocompatible. A mass transport limiting layer may provide many functions, e.g., biocompatibility and/or interferent-eliminating functions, etc. 
     In certain embodiments, a mass transport limiting layer is a membrane composed of crosslinked polymers containing heterocyclic nitrogen groups, such as polymers of polyvinylpyridine and polyvinylimidazole. Embodiments also include membranes that are made of a polyurethane, or polyether urethane, or chemically related material, or membranes that are made of silicone, and the like. 
     A membrane may be formed by crosslinking in situ a polymer, modified with a zwitterionic moiety, a non-pyridine copolymer component, and optionally another moiety that is either hydrophilic or hydrophobic, and/or has other desirable properties, in an alcohol-buffer solution. The modified polymer may be made from a precursor polymer containing heterocyclic nitrogen groups. For example, a precursor polymer may be polyvinylpyridine or polyvinylimidazole. Optionally, hydrophilic or hydrophobic modifiers may be used to “fine-tune” the permeability of the resulting membrane to an analyte of interest. Optional hydrophilic modifiers, such as poly(ethylene glycol), hydroxyl or polyhydroxyl modifiers, may be used to enhance the biocompatibility of the polymer or the resulting membrane. 
     A membrane may be formed in situ by applying an alcohol-buffer solution of a crosslinker and a modified polymer over an enzyme-containing sensing region and allowing the solution to cure for about one to two days or other appropriate time period. The crosslinker-polymer solution may be applied to the sensing region by placing a droplet or droplets of the membrane solution on the sensor, by dipping the sensor into the membrane solution, by spraying the membrane solution on the sensor, and the like. Generally, the thickness of the membrane is controlled by the concentration of the membrane solution, by the number of droplets of the membrane solution applied, by the number of times the sensor is dipped in the membrane solution, by the volume of membrane solution sprayed on the sensor, or by any combination of these factors. A membrane applied in this manner may have any combination of the following functions: (1) mass transport limitation, e.g., reduction of the flux of analyte that can reach the sensing region, (2) biocompatibility enhancement, or (3) interferent reduction. 
     In some instances, the membrane may form one or more bonds with the sensing region. By bonds is meant any type of an interaction between atoms or molecules that allows chemical compounds to form associations with each other, such as, but not limited to, covalent bonds, ionic bonds, dipole-dipole interactions, hydrogen bonds, London dispersion forces, and the like. For example, in situ polymerization of the membrane can form crosslinks between the polymers of the membrane and the polymers in the sensing region. In certain embodiments, crosslinking of the membrane to the sensing region facilitates a reduction in the occurrence of delamination of the membrane from the sensing region. 
     The substrate may be formed using a variety of non-conducting materials, including, for example, polymeric or plastic materials and ceramic materials. Suitable materials for a particular sensor may be determined, at least in part, based on the desired use of the sensor and properties of the materials. 
     In some embodiments, the substrate is flexible. For example, if the sensor is configured for implantation into a user, then the sensor may be made flexible (although rigid sensors may also be used for implantable sensors) to reduce pain to the user and damage to the tissue caused by the implantation of and/or the wearing of the sensor. A flexible substrate often increases the user&#39;s comfort and allows a wider range of activities. Suitable materials for a flexible substrate include, for example, non-conducting plastic or polymeric materials and other non-conducting, flexible, deformable materials. Examples of useful plastic or polymeric materials include thermoplastics such as polycarbonates, polyesters (e.g., Mylar™ and polyethylene terephthalate (PET)), polyvinyl chloride (PVC), polyurethanes, polyethers, polyamides, polyimides, or copolymers of these thermoplastics, such as PETG (glycol-modified polyethylene terephthalate). 
     In other embodiments, the sensors are made using a relatively rigid substrate to, for example, provide structural support against bending or breaking. Examples of rigid materials that may be used as the substrate include poorly conducting ceramics, such as aluminum oxide and silicon dioxide. An implantable sensor having a rigid substrate may have a sharp point and/or a sharp edge to aid in implantation of a sensor without an additional insertion device. 
     It will be appreciated that for many sensors and sensor applications, both rigid and flexible sensors will operate adequately. The flexibility of the sensor may also be controlled and varied along a continuum by changing, for example, the composition and/or thickness of the substrate. 
     In addition to considerations regarding flexibility, it is often desirable that implantable sensors should have a substrate which is physiologically harmless, for example, a substrate approved by a regulatory agency or private institution for in vivo use. 
     The sensor may include optional features to facilitate insertion of an implantable sensor. For example, the sensor may be pointed at the tip to ease insertion. In addition, the sensor may include a barb which assists in anchoring the sensor within the tissue of the user during operation of the sensor. However, the barb is typically small enough so that little damage is caused to the subcutaneous tissue when the sensor is removed for replacement. 
     An implantable sensor may also, optionally, have an anticlotting agent disposed on a portion of the substrate which is implanted into a user. This anticlotting agent may reduce or eliminate the clotting of blood or other body fluid around the sensor, particularly after insertion of the sensor. Blood clots may foul the sensor or irreproducibly reduce the amount of analyte which diffuses into the sensor. Examples of useful anticlotting agents include heparin and tissue plasminogen activator (TPA), as well as other known anticlotting agents. 
     The anticlotting agent may be applied to at least a portion of that part of the sensor that is to be implanted. The anticlotting agent may be applied, for example, by bath, spraying, brushing, or dipping, etc. The anticlotting agent is allowed to dry on the sensor. The anticlotting agent may be immobilized on the surface of the sensor or it may be allowed to diffuse away from the sensor surface. The quantities of anticlotting agent disposed on the sensor may be below the amounts typically used for treatment of medical conditions involving blood clots and, therefore, have only a limited, localized effect. 
       FIG. 11  shows an example in vivo-based analyte monitoring system  1100  in accordance with certain embodiments of the present disclosure. As shown, analyte monitoring system  1100  includes on body electronics  1110  electrically coupled to in vivo analyte sensor  1101  (a proximal portion of which is shown in  FIG. 11 ) and attached to adhesive layer  1140  for attachment on a skin surface on the body of a user. On body electronics  1110  includes on body housing  1119  that defines an interior compartment. Also shown in  FIG. 11  is insertion device  1150  that, when operated, transcutaneously positions a portion of analyte sensor  1101  through a skin surface and in fluid contact with bodily fluid, and positions on body electronics  1110  and adhesive layer  1140  on a skin surface. In certain embodiments, on body electronics  1110 , analyte sensor  1101  and adhesive layer  1140  are sealed within the housing of insertion device  1150  before use, and in certain embodiments, adhesive layer  1140  is also sealed within the housing or itself provides a terminal seal of the insertion device  1150 . 
     Referring back to the  FIG. 11 , analyte monitoring system  1100  includes display device  1120  which includes a display  1122  to output information to the user, an input component  1121  such as a button, actuator, a touch sensitive switch, a capacitive switch, pressure sensitive switch, jog wheel or the like, to input data or command to display device  1120  or otherwise control the operation of display device  1120 . It is noted that some embodiments may include display-less devices or devices without any user interface components. These devices may be functionalized to store data as a data logger and/or provide a conduit to transfer data from on body electronics and/or a display-less device to another device and/or location. Embodiments will be described herein as display devices for example purposes which are in no way intended to limit the embodiments of the present disclosure. It will be apparent that display-less devices may also be used in certain embodiments. 
     In certain embodiments, on body electronics  1110  may be configured to store some or all of the monitored analyte related data received from analyte sensor  1101  in a memory during the monitoring time period, and maintain it in memory until the usage period ends. In such embodiments, stored data is retrieved from on body electronics  1110  at the conclusion of the monitoring time period, for example, after removing analyte sensor  1101  from the user by detaching on body electronics  1110  from the skin surface where it was positioned during the monitoring time period. In such data logging configurations, real time monitored analyte level is not communicated to display device  1120  during the monitoring period or otherwise transmitted from on body electronics  1110 , but rather, retrieved from on body electronics  1110  after the monitoring time period. 
     In certain embodiments, input component  1121  of display device  1120  may include a microphone and display device  1120  may include software configured to analyze audio input received from the microphone, such that functions and operation of the display device  1120  may be controlled by voice commands. In certain embodiments, an output component of display device  1120  includes a speaker for outputting information as audible signals. Similar voice responsive components such as a speaker, microphone and software routines to generate, process and store voice driven signals may be provided to on body electronics  1110 . 
     In certain embodiments, display  1122  and input component  1121  may be integrated into a single component, for example a display that can detect the presence and location of a physical contact touch upon the display such as a touch screen user interface. In such embodiments, the user may control the operation of display device  1120  by utilizing a set of pre-programmed motion commands, including, but not limited to, single or double tapping the display, dragging a finger or instrument across the display, motioning multiple fingers or instruments toward one another, motioning multiple fingers or instruments away from one another, etc. In certain embodiments, a display includes a touch screen having areas of pixels with single or dual function capacitive elements that serve as LCD elements and touch sensors. 
     Display device  1120  also includes data communication port  1123  for wired data communication with external devices such as remote terminal (personal computer)  1170 , for example. Example embodiments of the data communication port  1123  include USB port, mini USB port, RS-232 port, Ethernet port, Firewire port, or other similar data communication ports configured to connect to the compatible data cables. Display device  1120  may also include an integrated in vitro glucose meter, including in vitro test strip port  1124  to receive an in vitro glucose test strip for performing in vitro blood glucose measurements. 
     Referring still to  FIG. 11 , display  1122  in certain embodiments is configured to display a variety of information—some or all of which may be displayed at the same or different time on display  1122 . In certain embodiments, the displayed information is user-selectable so that a user can customize the information shown on a given display screen. Display  1122  may include but is not limited to graphical display  1138 , for example, providing a graphical output of ketone values over a monitored time period (which may show important markers such as meals, exercise, sleep, heart rate, blood pressure, etc.), numerical display  1132 , for example, providing monitored ketone values (acquired or received in response to the request for the information), and trend or directional arrow display  1131  that indicates a rate of analyte change and/or a rate of the rate of analyte change. 
     As further shown in  FIG. 11 , display  1122  may also include date display  1135  providing for example, date information for the user, time of day information display  1139  providing time of day information to the user, battery level indicator display  1133  which graphically shows the condition of the battery (rechargeable or disposable) of the display device  1120 , sensor calibration status icon display  1134  for example, in monitoring systems that require periodic, routine or a predetermined number of user calibration events, notifying the user that the analyte sensor calibration is necessary, audio/vibratory settings icon display  1136  for displaying the status of the audio/vibratory output or alarm state, and wireless connectivity status icon display  1137  that provides indication of wireless communication connection with other devices such as on body electronics, data processing module  1160 , and/or remote terminal  1170 . As additionally shown in  FIG. 11 , display  1122  may further include simulated touch screen buttons  1140 ,  1141  for accessing menus, changing display graph output configurations or otherwise for controlling the operation of display device  1120 . 
     Referring back to  FIG. 11 , in certain embodiments, display  1122  of display device  1120  may be additionally, or instead of visual display, configured to output alarms notifications such as alarm and/or alert notifications, glucose values etc., which may be audible, tactile, or any combination thereof In one aspect, the display device  1120  may include other output components such as a speaker, vibratory output component and the like to provide audible and/or vibratory output indication to the user in addition to the visual output indication provided on display  1122 . 
     After the positioning of on body electronics  1110  on the skin surface and analyte sensor  1101  in vivo to establish fluid contact with interstitial fluid (or other appropriate bodily fluid), on body electronics  1110  in certain embodiments is configured to wirelessly communicate analyte related data (such as, for example, data corresponding to monitored analyte level and/or monitored temperature data, and/or stored historical analyte related data) when on body electronics  1110  receives a command or request signal from display device  1120 . In certain embodiments, on body electronics  1110  may be configured to at least periodically broadcast real time data associated with monitored analyte level which is received by display device  1120  when display device  1120  is within communication range of the data broadcast from on body electronics  1110 , e.g., it does not need a command or request from a display device to send information. 
     For example, display device  1120  may be configured to transmit one or more commands to on body electronics  1110  to initiate data transfer, and in response, on body electronics  1110  may be configured to wirelessly transmit stored analyte related data collected during the monitoring time period to display device  1120 . Display device  1120  may in turn be connected to a remote terminal  1170  such as a personal computer and functions as a data conduit to transfer the stored analyte level information from the on body electronics  1110  to remote terminal  1170 . In certain embodiments, the received data from the on body electronics  1110  may be stored (permanently or temporarily) in one or more memory of the display device  1120 . In certain other embodiments, display device  1120  is configured as a data conduit to pass the data received from on body electronics  1110  to remote terminal  1170  that is connected to display device  1120 . 
     Referring still to  FIG. 11 , also shown in analyte monitoring system  1100  are data processing module  1160  and remote terminal  1170 . Remote terminal  1170  may include a personal computer, a server terminal a laptop computer or other suitable data processing devices including software for data management and analysis and communication with the components in the analyte monitoring system  1100 . For example, remote terminal  1170  may be connected to a local area network (LAN), a wide area network (WAN), or other data network for uni-directional or bi-directional data communication between remote terminal  1170  and display device  1120  and/or data processing module  1160 . 
     Remote terminal  1170  in certain embodiments may include one or more computer terminals located at a physician&#39;s office or a hospital. For example, remote terminal  1170  may be located at a location other than the location of display device  1120 . Remote terminal  1170  and display device  1120  could be in different rooms or different buildings. Remote terminal  1170  and display device  1120  could be at least about one mile apart, e.g., at least about 10 miles apart, e.g., at least about 1100 miles apart. For example, remote terminal  1170  could be in the same city as display device  1120 , remote terminal  1170  could be in a different city than display device  1120 , remote terminal  1170  could be in the same state as display device  1120 , remote terminal  1170  could be in a different state than display device  1120 , remote terminal  1170  could be in the same country as display device  1120 , or remote terminal  1170  could be in a different country than display device  1120 , for example. 
     In certain embodiments, a separate, optional data communication/processing device such as data processing module  1160  may be provided in analyte monitoring system  1100 . Data processing module  1160  may include components to communicate using one or more wireless communication protocols such as, for example, but not limited to, infrared (IR) protocol, Bluetooth protocol, Zigbee protocol, and 802.11 wireless LAN protocol. Additional description of communication protocols including those based on Bluetooth protocol and/or Zigbee protocol can be found in U.S. Patent Publication No. 2006/0193375 incorporated herein by reference in its entirety for all purposes. Data processing module  1160  may further include communication ports, drivers or connectors to establish wired communication with one or more of display device  1120 , on body electronics  1110 , or remote terminal  1170  including, for example, but not limited to USB connector and/or USB port, Ethernet connector and/or port, FireWire connector and/or port, or RS-232 port and/or connector. 
     In certain embodiments, data processing module  1160  is programmed to transmit a polling or query signal to on body electronics  1110  at a predetermined time interval (e.g., once every minute, once every five minutes, or the like), and in response, receive the monitored analyte level information from on body electronics  1110 . Data processing module  1160  stores in its memory the received analyte level information, and/or relays or retransmits the received information to another device such as display device  1120 . More specifically in certain embodiments, data processing module  1160  may be configured as a data relay device to retransmit or pass through the received analyte level data from on body electronics  1110  to display device  1120  or a remote terminal (for example, over a data network such as a cellular or WiFi data network) or both. 
     In certain embodiments, on body electronics  1110  and data processing module  1160  may be positioned on the skin surface of the user within a predetermined distance of each other (for example, about 1-12 inches, or about 1-10 inches, or about 1-7 inches, or about 1-5 inches) such that periodic communication between on body electronics  1110  and data processing module  1160  is maintained. Alternatively, data processing module  1160  may be worn on a belt or clothing item of the user, such that the desired distance for communication between the on body electronics  1110  and data processing module  1160  for data communication is maintained. In a further aspect, the housing of data processing module  1160  may be configured to couple to or engage with on body electronics  1110  such that the two devices are combined or integrated as a single assembly and positioned on the skin surface. In further embodiments, data processing module  1160  is detachably engaged or connected to on body electronics  1110  providing additional modularity such that data processing module  1160  may be optionally removed or reattached as desired. 
     Referring again to  FIG. 11 , in certain embodiments, data processing module  1160  is programmed to transmit a command or signal to on body electronics  1110  at a predetermined time interval such as once every minute, or once every 5 minutes or once every 30 minutes or any other suitable or desired programmable time interval to request analyte related data from on body electronics  1110 . When data processing module  1160  receives the requested analyte related data, it stores the received data. In this manner, analyte monitoring system  1100  may be configured to receive the continuously monitored analyte related information at the programmed or programmable time interval, which is stored and/or displayed to the user. The stored data in data processing module  1160  may be subsequently provided or transmitted to display device  1120 , remote terminal  1170  or the like for subsequent data analysis such as identifying frequency of periods of glycemic level excursions over the monitored time period, or the frequency of the alarm event occurrence during the monitored time period, for example, to improve therapy related decisions. Using this information, the doctor, healthcare provider or the user may adjust or recommend modification to the diet, daily habits and routines such as exercise, and the like. 
     In another embodiment, data processing module  1160  transmits a command or signal to on body electronics  1110  to receive the analyte related data in response to a user activation of a switch provided on data processing module  1160  or a user initiated command received from display device  1120 . In further embodiments, data processing module  1160  is configured to transmit a command or signal to on body electronics  1110  in response to receiving a user initiated command only after a predetermined time interval has elapsed. For example, in certain embodiments, if the user does not initiate communication within a programmed time period, such as, for example about 5 hours from last communication (or 10 hours from the last communication, or 24 hours from the last communication), the data processing module  1160  may be programmed to automatically transmit a request command or signal to on body electronics  1110 . Alternatively, data processing module  1160  may be programmed to activate an alarm to notify the user that a predetermined time period of time has elapsed since the last communication between the data processing module  1160  and on body electronics  1110 . In this manner, users or healthcare providers may program or configure data processing module  1160  to provide certain compliance with analyte monitoring regimen, so that frequent determination of analyte levels is maintained or performed by the user. 
     In certain embodiments, when a programmed or programmable alarm condition is detected (for example, a detected glucose level monitored by analyte sensor  1101  that is outside a predetermined acceptable range indicating a physiological condition which requires attention or intervention for medical treatment or analysis (for example, ketosis, diabetic ketoacidosis, an impending ketosisor an impending diabetic ketoacidosis), the one or more output indications may be generated by the control logic or processor of the on body electronics  1110  and output to the user on a user interface of on body electronics  1110  so that corrective action may be timely taken. In addition to or alternatively, if display device  1120  is within communication range, the output indications or alarm data may be communicated to display device  1120  whose processor, upon detection of the alarm data reception, controls the display  1122  to output one or more notification. 
     In certain embodiments, control logic or processors of on body electronics  1110  can execute software programs stored in memory to determine future or anticipated analyte levels based on information obtained from analyte sensor  1101 , e.g., the current analyte level, the rate of change of the analyte level, the acceleration of the analyte level change, and/or analyte trend information determined based on stored monitored analyte data providing a historical trend or direction of analyte level fluctuation as function time during monitored time period. Predictive alarm parameters may be programmed or programmable in display device  1120 , or the on body electronics  1110 , or both, and output to the user in advance of anticipating the user&#39;s analyte level reaching the future level. This provides the user an opportunity to take timely corrective action. 
     Information, such as variation or fluctuation of the monitored analyte level as a function of time over the monitored time period providing analyte trend information, for example, may be determined by one or more control logic or processors of display device  1120 , data processing module  1160 , and/or remote terminal  1170 , and/or on body electronics  1110 . Such information may be displayed as, for example, a graph (such as a line graph) to indicate to the user the current and/or historical and/or and predicted future analyte levels as measured and predicted by the analyte monitoring system  1100 . Such information may also be displayed as directional arrows (for example, see trend or directional arrow display  1131 ) or other icon(s), e.g., the position of which on the screen relative to a reference point indicated whether the analyte level is increasing or decreasing as well as the acceleration or deceleration of the increase or decrease in analyte level. This information may be utilized by the user to determine any necessary corrective actions to ensure the analyte level remains within an acceptable and/or clinically safe range. Other visual indicators, including colors, flashing, fading, etc., as well as audio indicators including a change in pitch, volume, or tone of an audio output and/or vibratory or other tactile indicators may also be incorporated into the display of trend data as means of notifying the user of the current level and/or direction and/or rate of change of the monitored analyte level. For example, based on a determined rate of glucose change, programmed clinically significant glucose threshold levels (e.g., hyperglycemic and/or hypoglycemic levels), and current analyte level derived by an in vivo analyte sensor, the system  1100  may include an algorithm stored on computer readable medium to determine the time it will take to reach a clinically significant level and will output notification in advance of reaching the clinically significant level, e.g., 30 minutes before a clinically significant level is anticipated, and/or 20 minutes, and/or 10 minutes, and/or 5 minutes, and/or 3 minutes, and/or 1 minute, and so on, with outputs increasing in intensity or the like. 
     Referring again back to  FIG. 11 , in certain embodiments, software algorithm(s) for execution by data processing module  1160  may be stored in an external memory device such as an SD card, microSD card, compact flash card, XD card, Memory Stick card, Memory Stick Duo card, or USB memory stick/device including executable programs stored in such devices for execution upon connection to the respective one or more of the on body electronics  1110 , remote terminal  1170  or display device  1120 . In a further aspect, software algorithms for execution by data processing module  1160  may be provided to a communication device such as a mobile telephone including, for example, WiFi or Internet enabled smart phones or personal digital assistants (PDAs) as a downloadable application for execution by the downloading communication device. 
     Examples of smart phones include Windows®, Android™, iPhone® operating system, Palm® WebOS™, Blackberry® operating system, or Symbian® operating system based mobile telephones with data network connectivity functionality for data communication over an internet connection and/or a local area network (LAN). PDAs as described above include, for example, portable electronic devices including one or more processors and data communication capability with a user interface (e.g., display/output unit and/or input unit, and configured for performing data processing, data upload/download over the internet, for example. In such embodiments, remote terminal  1170  may be configured to provide the executable application software to the one or more of the communication devices described above when communication between the remote terminal  1170  and the devices are established. 
     On Body Electronics 
     In certain embodiments, on body electronics (or sensor control device)  1110  ( FIG. 11 ) includes at least a portion of the electronic components that operate the sensor and the display device. The electronic components of the on body electronics typically include a power supply for operating the on body electronics and the sensor, a sensor circuit for obtaining signals from and operating the sensor, a measurement circuit that converts sensor signals to a desired format, and a processing circuit (or processing circuitry) that, at minimum, obtains signals from the sensor circuit and/or measurement circuit and provides the signals to an optional on body electronics. In some embodiments, the processing circuit may also partially or completely evaluate the signals from the sensor and convey the resulting data to the optional on body electronics and/or activate an optional alarm system if the analyte level exceeds a threshold. The processing circuit often includes digital logic circuitry. 
     The on body electronics may optionally contain electronics for transmitting the sensor signals or processed data from the processing circuit to a receiver/display unit; a data storage unit for temporarily or permanently storing data from the processing circuit; a temperature probe circuit for receiving signals from and operating a temperature probe; a reference voltage generator for providing a reference voltage for comparison with sensor-generated signals; and/or a watchdog circuit that monitors the operation of the electronic components in the on body electronics. 
     Moreover, the on body electronics may also include digital and/or analog components utilizing semiconductor devices, including transistors. To operate these semiconductor devices, the on body electronics may include other components including, for example, a bias control generator to correctly bias analog and digital semiconductor devices, an oscillator to provide a clock signal, and a digital logic and timing component to provide timing signals and logic operations for the digital components of the circuit. 
     As an example of the operation of these components, the sensor circuit and the optional temperature probe circuit provide raw signals from the sensor to the measurement circuit. The measurement circuit converts the raw signals to a desired format, using for example, a current-to-voltage converter, current-to-frequency converter, and/or a binary counter or other indicator that produces a signal proportional to the absolute value of the raw signal. This may be used, for example, to convert the raw signal to a format that can be used by digital logic circuits. The processing circuit may then, optionally, evaluate the data and provide commands to operate the electronics. 
       FIG. 12  is a block diagram of the on body electronics  1110  ( FIG. 11 ) in certain embodiments. Referring to  FIG. 12 , on body electronics  1110  in certain embodiments includes a control unit  1210  (such as, for example but not limited to, one or more processors (or processing circuitry) and/or ASICs with processing circuitry), operatively coupled to analog front end circuitry  1270  to process signals such as raw current signals received from analyte sensor  1101 . Also shown in  FIG. 12  is memory  1220  operatively coupled to control unit  1210  for storing data and/or software routines for execution by control unit  1210 . Memory  1220  in certain embodiments may include electrically erasable programmable read only memory (EEPROM), erasable programmable read only memory (EPROM), random access memory (RAM), read only memory (ROM), flash memory, or one or more combinations thereof. 
     In certain embodiments, control unit  1210  accesses data or software routines stored in the memory  1220  to update, store or replace stored data or information in the memory  1220 , in addition to retrieving one or more stored software routines for execution. Also shown in  FIG. 12  is power supply  1260  which, in certain embodiments, provides power to some or all of the components of on body electronics  1110 . For example, in certain embodiments, power supply  1260  is configured to provide power to the components of on body electronics  1110  except for communication module  1240 . In such embodiments, on body electronics  1110  is configured to operate analyte sensor  1101  to detect and monitor the analyte level at a predetermined or programmed (or programmable) time intervals, and generating and storing, for example, the signals or data corresponding to the detected analyte levels. 
     In certain embodiments, power supply  1260  in on body electronics  1110  may be toggled between its internal power source (e.g., a battery) and the RF power received from display device  1120 . For example, in certain embodiments, on body electronics  1110  may include a diode or a switch that is provided in the internal power source connection path in on body electronics  1110  such that, when a predetermined level of RF power is detected by on body electronics  1110 , the diode or switch is triggered to disable the internal power source connection (e.g., making an open circuit at the power source connection path), and the components of on body electronics is powered with the received RF power. The open circuit at the power source connection path prevents the internal power source from draining or dissipating as in the case when it is used to power on body electronics  1110 . 
     When the RF power from display device  1120  falls below the predetermined level, the diode or switch is triggered to establish the connection between the internal power source and the other components of on body electronics  1110  to power the on body electronics  1110  with the internal power source. In this manner, in certain embodiments, toggling between the internal power source and the RF power from display device  1120  may be configured to prolong or extend the useful life of the internal power source. 
     The stored analyte related data, however, is not transmitted or otherwise communicated to another device such as display device  1120  ( FIG. 11 ) until communication module  1240  is separately powered, for example, with the RF power from display device  1120  that is positioned within a predetermined distance from on body electronics  1110 . In such embodiments, analyte level is sampled based on the predetermined or programmed time intervals as discussed above, and stored in memory  1220 . When analyte level information is requested, for example, based on a request or transmit command received from another device such as display device  1120  ( FIG. 11 ), using the RF power from the display device, communication module  1240  of on body electronics  1110  initiates data transfer to the display device  1120 . 
     Referring back to  FIG. 12 , an optional output unit  1250  is provided to on body electronics  1110 . In certain embodiments, output unit  1250  may include an LED indicator, for example, to alert the user of one or more predetermined conditions associated with the operation of the on body electronics  1110  and/or the determined analyte level. By way of nonlimiting example, the on body electronics  1110  may be programmed to assert a notification using an LED indicator, or other indicator on the on body electronics  1110  when signals (based on one sampled sensor data point, or multiple sensor data points) received from analyte sensor  1101  are indicated to be beyond a programmed acceptable range, potentially indicating a health risk condition such as hyperglycemia or hypoglycemia, or the onset or potential of such conditions. With such prompt or indication, the user may be timely informed of such potential condition, and using display device  1120 , acquire the glucose level information from the on body electronics  1110  to confirm the presence of such conditions so that timely corrective actions may be taken. 
     Referring again to  FIG. 12 , antenna  1230  and communication module  1240  operatively coupled to the control unit  1210  may be configured to detect and process the RF power when on body electronics  1110  is positioned within predetermined proximity to the display device  1120  ( FIG. 11 ) that is providing or radiating the RF power. Further, on body electronics  1110  may provide analyte level information and optionally analyte trend or historical information based on stored analyte level data, to display device  1120 . In certain aspects, the trend information may include a plurality of analyte level information over a predetermined time period that are stored in the memory  1220  of the on body electronics  1110  and provided to the display device  1120  with the real time analyte level information. For example, the trend information may include a series of time spaced analyte level data for the time period since the last transmission of the analyte level information to the display device  1120 . Alternatively, the trend information may include analyte level data for the prior 30 minutes or one hour that are stored in memory  1220  and retrieved under the control of the control unit  1210  for transmission to the display device  1120 . 
     In certain embodiments, on body electronics  1110  is configured to store analyte level data in first and second FIFO buffers that are part of memory  1220 . The first FIFO buffer stores 16 (or 10 or 20) of the most recent analyte level data spaced one minute apart. The second FIFO buffer stores the most recent 8 hours (or 10 hours or 3 hours) of analyte level data spaced 10 minutes (or 15 minutes or 20 minutes). The stored analyte level data are transmitted from on body electronics  1110  to display unit  1120  in response to a request received from display unit  1120 . Display unit  1120  uses the analyte level data from the first FIFO buffer to estimate glucose rate-of-change and analyte level data from the second FIFO buffer to determine historical plots or trend information. 
     In certain embodiments, for configurations of the on body electronics that includes a power supply, the on body electronics may be configured to detect an RF control command (ping signal) from the display device  1120 . More specifically, an On/Off Key (OOK) detector may be provided in the on body electronics which is turned on and powered by the power supply of the on body electronics to detect the RF control command or the ping signal from the display device  1120 . Additional details of the OOK detector are provided in U.S. Patent Publication No. 2008/0278333, the disclosure of which is incorporated by reference in its entirety for all purposes. In certain aspects, when the RF control command is detected, on body electronics determines what response packet is necessary, and generates the response packet for transmission back to the display device  1120 . In this embodiment, the analyte sensor  1101  continuously receives power from the power supply or the battery of the on body electronics and operates to monitor the analyte level continuously in use. However, the sampled signal from the analyte sensor  1101  may not be provided to the display device  1120  until the on body electronics receives the RF power (from the display device  1120 ) to initiate the transmission of the data to the display device  1120 . In one embodiment, the power supply of the on body electronics may include a rechargeable battery which charges when the on body electronics receives the RF power (from the display device  1120 , for example). 
     Referring back to  FIG. 11 , in certain embodiments, on body electronics  1110  and the display device  1120  may be configured to communicate using RFID (radio frequency identification) protocols. More particularly, in certain embodiments, the display device  1120  is configured to interrogate the on body electronics  1110  (associated with an RFID tag) over an RF communication link, and in response to the RF interrogation signal from the display device  1120 , on body electronics  1110  provides an RF response signal including, for example, data associated with the sampled analyte level from the sensor  1101 . Additional information regarding the operation of RFID communication can be found in U.S. Pat. No. 7,545,272, and in U.S. application Ser. Nos. 12/698,624, 12/699,653, 12/761,387, and U.S. Patent Publication No. 2009/0108992 the disclosures of all of which are incorporated herein by reference in their entireties and for all purposes. 
     For example, in one embodiment, the display device  1120  may include a backscatter RFID reader configured to provide an RF field such that when on body electronics  1110  is within the transmitted RF field of the RFID reader, on body electronics  1110  antenna is tuned and in turn provides a reflected or response signal (for example, a backscatter signal) to the display device  1120 . The reflected or response signal may include sampled analyte level data from the analyte sensor  1101 . 
     In certain embodiments, when display device  1120  is positioned in within a predetermined range of the on body electronics  1110  and receives the response signal from the on body electronics  1110 , the display device  1120  is configured to output an indication (audible, visual or otherwise) to confirm the analyte level measurement acquisition. That is, during the course of the 5 to 10 days of wearing the on body electronics  1110 , the user may at any time position the display device  1120  within a predetermined distance (for example, about 1-5 inches, or about 1-10 inches, or about 1-12 inches) from on body electronics  1110 , and after waiting a few seconds of sample acquisition time period, an audible indication is output confirming the receipt of the real time analyte level information. The received analyte information may be output to the display  1122  ( FIG. 11 ) of the display device  1120  for presentation to the user. 
     Display Devices 
       FIG. 13  is a block diagram of display device  1120  as shown in  FIG. 11  in certain embodiments. Although the term display device is used, the device can be configured to read without displaying data, and can be provided without a display, such as can be the case with a relay or other device that relays a received signal according to the same or a different transmission protocol (e.g., NFC-to-Bluetooth or Bluetooth Low Energy). Referring to  FIG. 13 , display device  1120  ( FIG. 11 ) includes control unit  1310 , such as one or more processors (or processing circuitry) operatively coupled to a display  1122 , and an input component (e.g., user interface)  1121 . The display device  1120  may also include one or more data communication ports such as USB port (or connector)  1123  or RS-232 port  1330  (or any other wired communication ports) for data communication with a data processing module  1160  ( FIG. 11 ), remote terminal  1170  ( FIG. 11 ), or other devices such as a personal computer, a server, a mobile computing device, a mobile telephone, a pager, or other handheld data processing devices including mobile telephones such as internet connectivity enabled smart phones, with data communication and processing capabilities including data storage and output. 
     Referring back to  FIG. 13 , display device  1120  may include a strip port  1124  configured to receive in vitro test strips, the strip port  1124  coupled to the control unit  1310 , and further, where the control unit  1310  includes programming to process the sample on the in vitro test strip which is received in the strip port  1124 . Any suitable in vitro test strip may be employed, e.g., test strips that only require a very small amount (e.g., one microliter or less, e.g., about 0.5 microliter or less, e.g., about 0.1 microliter or less), of applied sample to the strip in order to obtain accurate glucose information. Display devices with integrated in vitro monitors and test strip ports may be configured to conduct in vitro analyte monitoring with no user calibration of the in vitro test strips (e.g., no human intervention calibration). 
     In certain embodiments, an integrated in vitro meter can accept and process a variety of different types of test strips (e.g., those that require user calibration and those that do not), some of which may use different technologies (those that operate using amperometric techniques and those that operate using coulometric techniques), etc. Detailed description of such test strips and devices for conducting in vitro analyte monitoring is provided in U.S. Pat. Nos. 6,377,894, 6,616,819, 7,749,740, 7,418,285; U.S. Patent Publication Nos. 2004/0118704, 2006/0096006, 2008/0066305, 2008/0267823, 2010/0094610, 2010/0094111, and 2010/0094112, and U.S. application Ser. No. 12/695,947, the disclosures of all of which are incorporated herein by reference in their entireties and for all purposes. 
     Ketone information obtained by the in vitro glucose testing device may be used for a variety of purposes. For example, the information may be used to confirm results of analyte sensor  1101  to increase the confidence in the results from sensor  1101  indicating the monitored analyte level (e.g., in instances in which information obtained by sensor  1101  is employed in therapy related decisions), etc. In certain embodiments, analyte sensors do not require calibration by human intervention during its usage life. However, in certain embodiments, a system may be programmed to self-detect problems and take action, e.g., shut off and/or notify a user. For example, an analyte monitoring system may be configured to detect system malfunction, or potential degradation of sensor stability or potential adverse condition associated with the operation of the analyte sensor, the system may notify the user, using display device  1120  ( FIG. 11 ) for example, to perform analyte sensor calibration or compare the results received from the analyte sensor corresponding to the monitored analyte level, to a reference value (such as a result from an in vitro blood glucose measurement). 
     In certain embodiments, when the potential adverse condition associated with the operation of the sensor, and/or potential sensor stability degradation condition is detected, the system may be configured to shut down (automatically without notification to the user, or after notifying the user) or disable the output or display of the monitored analyte level information received the on body electronics assembly. In certain embodiments, the analyte monitoring system may be shut down or disabled temporarily to provide an opportunity to the user to correct any detected adverse condition or sensor instability. In certain other embodiments, the analyte monitoring system may be permanently disabled when the adverse sensor operation condition or sensor instability is detected. 
     Referring still to  FIG. 13 , power supply  1320 , such as one or more batteries, rechargeable or single use disposable, is also provided and operatively coupled to control unit  1310 , and configured to provide the necessary power to display device  1120  ( FIG. 11 ) for operation. In addition, display device  1120  may include an antenna  1351  such as a 433 MHz (or other equivalent) loop antenna, 13.56 MHz antenna, or a 2.45 G Hz antenna, coupled to a receiver processor  1350  (which may include a 433 MHz, 13.56 MHz, or 2.45 GHz transceiver chip, for example) for wireless communication with the on body electronics  1110  ( FIG. 11 ). Additionally, an inductive loop antenna  1341  is provided and coupled to a squarewave driver  1340  which is operatively coupled to control unit  1310 . 
     In certain embodiments, data packets received from on body electronics and received in response to a request from display device, for example, include one or more of a current glucose level from the analyte sensor, a current estimated rate of glycemic change, and a glucose trend history based on automatic readings acquired and stored in memory of on skin electronics. For example, current glucose level may be output on display  1122  of display device  1120  as a numerical value, the current estimated rage of glycemic change may be output on display  1122  as a directional arrow  1131  ( FIG. 11 ), and glucose trend history based on stored monitored values may be output on display  1122  as a graphical trace  1138  ( FIG. 11 ). In certain embodiments, the processor (or processing circuitry) of display device  1120  may be programmed to output more or less information for display on display  1122 , and further, the type and amount of information output on display  1122  may be programmed or programmable by the user. 
     Data Communication and Processing Routines 
     Referring now to  FIG. 14  which illustrates data and/or commands exchange between on body electronics  1110  and display device  1120  during the initialization and pairing routine, display device  1120  provides and initial signal  1421  to on body electronics  1110 . When the received initial signal  1421  includes RF energy exceeding a predetermined threshold level  1403 , an envelope detector of on body electronics  1110  is triggered  1404 , one or more oscillators of on body electronics  1110  turns on, and control logic or processors of on body electronics  1110  is temporarily latched on to retrieve and execute one or more software routines to extract the data stream from the envelope detector  1404 . If the data stream from the envelope detector returns a valid query  1405 , a reply signal  1422  is transmitted to display device  1120 . The reply signal  1422  from on body electronics  1110  includes an identification code such as on body electronics  1110  serial number. Thereafter, the on body electronics  1110  returns to shelf mode in an inactive state. 
     On the other hand, if the data stream from the envelope detector does not return a valid query from display device  1120 , on body electronics  1110  does not transmit a reply signal to display device  1120  nor is on body electronics  1110  serial number provided to display device  1120 . Thereafter, on body electronics  1110  returns to shelf mode  1403 , and remains in powered down state until it detects a subsequent initial signal  1421  from display device  1120 . 
     When display device  1120  receives the data packet including identification information or serial number from on body electronics  1110 , it extracts that information from the data packet  1412 . With the extracted on body electronics  1110  serial number, display device  1120  determines whether on body electronics  1110  associated with the received serial number is configured. If on body electronics  1110  associated with the received serial number has already been configured, for example, by another display device, display device  1120  returns to the beginning of the routine to transmit another initial signal  1411  in an attempt to initialize another on body electronics that has not been configured yet. In this manner, in certain embodiments, display device  1120  is configured to pair with an on body electronics that has not already been paired with or configured by another display device. 
     Referring back to  FIG. 14 , if on body electronics  1110  associated with the extracted serial number has not been configured  1413 , display device  1120  is configured to transmit a wake up signal to on body electronics  1110  which includes a configure command. In certain embodiments, wake up command from display device  1120  includes a serial number of on body electronics  1110  so that only the on body electronics with the same serial number included in the wake up command detects and exits the inactive shelf mode and enters the active mode. More specifically, when the wake up command including the serial number is received by on body electronics  1110 , control logic or one or more processors (or processing circuitry) of on body electronics  1110  executes routines  1403 ,  1404 , and  1405  to temporarily exit the shelf mode, when the RF energy received with the wakeup signal (including the configure command) exceeds the threshold level, and determines that it is not a valid query (as that determination was previously made and its serial number transmitted to display device  1120 ). Thereafter, on body electronics  1110  determines whether the received serial number (which was received with the wake up command) matches its own stored serial number  1406 . If the two serial numbers do not match, routine returns to the beginning where on body electronics  1110  is again placed in inactive shelf mode  1402 . On the other hand, if on body electronics  1110  determines that the received serial number matches its stored serial number  1406 , control logic or one or more processors of on body electronics  1110  permanently latches on  1407 , and oscillators are turned on to activate on body electronics  1110 . Further, referring back to FIG,  14 , when on body electronics  1110  determines that the received serial number matches its own serial number  1406 , display device  1120  and on body electronics  1110  are successfully paired  1416 . 
     In this manner, using a wireless signal to turn on and initialize on body electronics  1110 , the shelf life of on body electronics  1110  may be prolonged since very little current is drawn or dissipated from on body electronics  1110  power supply during the time period that on body electronics  1110  is in inactive, shelf mode prior to operation. In certain embodiments, during the inactive shelf mode, on body electronics  1110  has minimal operation, if any, that require extremely low current. The RF envelope detector of on body electronics  1110  may operate in two modes—a desensitized mode where it is responsive to received signals of less than about 1 inch, and normal operating mode with normal signal sensitivity such that it is responsive to receives signals at a distance of about 3-12 inches. 
     During the initial pairing between display device  1120  and on body electronics  1110 , in certain embodiments, display device  1120  sends its identification information such as, for example, 4 bytes of display device ID which may include its serial number. On body electronics  1110  stores the received display device ID in one or more storage unit or memory component and subsequently includes the stored display device ID data in response packets or data provided to the display device  1120 . In this manner, display device  1120  can discriminate detected data packets from on body electronics  1110  to determine that the received or detected data packets originated from the paired or correct on body electronics  1110 . The pairing routine based on the display device ID in certain embodiments avoids potential collision between multiple devices, especially in the cases where on body electronics  1110  does not selectively provide the analyte related data to a particular display device, but rather, provide to any display device within range and/or broadcast the data packet to any display device in communication range. 
     In certain embodiments, the payload size from display device  1120  to on body electronics  1110  is 12 bytes, which includes 4 bytes of display device ID, 4 bytes of on body device ID, one byte of command data, one byte of spare data space, and two bytes for CRC (cyclic redundancy check) for error detection. 
     After pairing is complete, when display device  1120  queries on body electronics  1110  for real time monitored analyte information and/or logged or stored analyte data, in certain embodiments, the responsive data packet transmitted to display device  1120  includes a total of  418  bytes that includes 34 bytes of status information, time information and calibration data, 96 bytes of the most recent 16 one-minute glucose data points, and 288 bytes of the most recent 15 minute interval glucose data over the 12 hour period. Depending upon the size or capacity of the memory or storage unit of on body electronics  1110 , data stored and subsequently provided to the display device  1120  may have a different time resolution and/or span a longer or shorter time period. For example, with a larger data buffer, glucose related data provided to the display device  1120  may include glucose data over a 24 hour time period at 15 minute sampling intervals, 10 minute sampling intervals, 5 minute sampling intervals, or one minute sampling interval. Further, the determined variation in the monitored analyte level illustrating historical trend of the monitored analyte level may be processed and/or determined by the on body electronics  1110 , or alternatively or in addition to, the stored data may be provided to the display device  1120  which may then determine the trend information of the monitored analyte level based on the received data packets. 
     The size of the data packets provided to display device  1120  from on body electronics  1110  may also vary depending upon the communication protocol and/or the underlying data transmission frequency—whether using a 433 MHz, a 13.56 MHz, or 2.45 GHz in addition to other parameters such as, for example, the presence of data processing devices such as a processor or processing circuitry (e.g., central processing unit CPU) in on body electronics  1110 , in addition to the ASIC state machine, size of the data buffer and/or memory, and the like. 
     In certain embodiments, upon successful activation of on body electronics  1110  and pairing with display device  1120 , control unit of display device  1120  may be programmed to generate and output one or more visual, audible and/or haptic notifications to output to the user on display  1122 , or on the user interface of display device  1120 . In certain embodiments, only one display device can pair with one on body electronics at one time. Alternatively, in certain embodiments, one display device may be configured to pair with multiple on body electronics at the same time. 
     Once paired, display  1122  of display device  1120 , for example, outputs, under the control of the processor of display device  1120 , the remaining operational life of the analyte sensor  1101  in user. Furthermore, as the end of sensor life approaches, display device may be configured to output notifications to alert the user of the approaching end of sensor life. The schedule for such notification may be programmed or programmable by the user and executed by the processor of the display device. 
     Referring back to  FIG. 11 , in certain embodiments, analyte monitoring system  1100  may store the historical analyte data along with a date and/or time stamp and/or and contemporaneous temperature measurement, in memory, such as a memory configured as a data logger as described above. In certain embodiments, analyte data is stored at the frequency of about once per minute, or about once every ten minutes, or about once an hour, etc. Data logger embodiments may store historical analyte data for a predetermined period of time, e.g., a duration specified by a physician, for example, e.g., about 1 day to about 1 month or more, e.g., about 3 days or more, e.g., about 5 days or more, e.g., about 7 days or more, e.g., about 2 weeks or more, e.g., about 1 month or more. 
     Other durations of time may be suitable, depending on the clinical significance of the data being observed. The analyte monitoring system  1100  may display the analyte readings to the subject during the monitoring period. In some embodiments, no data is displayed to the subject. Optionally, the data logger can transmit the historical analyte data to a receiving device disposed adjacent, e.g., in close proximity to the data logger. For example, a receiving device may be configured to communicate with the data logger using a transmission protocol operative at low power over distances of a fraction of an inch to about several feet. For example, and without limitation, such close proximity protocols include Certified Wireless USB™, TransferJet™, Bluetooth® (IEEE 802.15.1), WiFi™ (IEEE 802.11), ZigBee® (IEEE 802.15.4-2006), Wibree™, or the like. 
     The analyte data parameters may be computed by a processor or processing circuitry executing a program stored in a memory. In certain embodiments, the processor executing the program stored in the memory is provided in data processing module  1160  ( FIG. 11 ). In certain embodiments, the processor executing the program stored in the memory is provided in display device  1120 . An example technique for analyzing data is the applied ambulatory glucose profile (AGP) analysis technique. Additional detailed descriptions are provided in U.S. Pat. Nos. 5,262,035; 5,264,104; 5,262,305; 5,320,715; 5,593,852; 6,175,752; 6,650,471; 6,746, 582, 6,284,478, 7,299,082, and in U.S. patent application Ser. Nos. 10/745,878; 11/060,365, the disclosures of all of which are incorporated herein by reference in their entireties for all purposes. 
     As described above, in certain aspects of the present disclosure, discrete ketone measurement data may be acquired on-demand or upon request from the display device, where the ketone measurement is obtained from an in vivo ketone sensor transcutaneously positioned under the skin layer of a user, and further having a portion of the sensor maintained in fluid contact with the bodily fluid under the skin layer. Accordingly, in aspects of the present disclosure, the user of the analyte monitoring system may conveniently determine real time glucose information at any time, using the RFID communication protocol as described above. 
     In one aspect, the integrated assembly including the on body electronics and the insertion device may be sterilized and packaged as one single device and provided to the user. Furthermore, during manufacturing, the insertion device assembly may be terminal packaged providing cost savings and avoiding the use of, for example, costly thermoformed tray or foil seal. In addition, the insertion device may include an end cap that is rotatably coupled to the insertion device body, and which provides a safe and sterile environment (and avoid the use of desiccants for the sensor) for the sensor provided within the insertion device along with the integrated assembly. Also, the insertion device sealed with the end cap may be configured to retain the sensor within the housing from significant movement during shipping such that the sensor position relative to the integrated assembly and the insertion device is maintained from manufacturing, assembly and shipping, until the device is ready for use by the user. 
     Example Embodiments of Ketone Sensors 
     The present disclosure discloses enzyme compositions that include nicotinamide adenine dinucleotide phosphate (NAD(P)+) or derivative thereof and an electron transfer agent having a transition metal complex. In some embodiments, the subject enzyme compositions include nicotinamide adenine dinucleotide phosphate (NAD(P)+) or derivative thereof, an NAD(P)+-dependent dehydrogenase, an NAD(P)H oxidoreductase and an electron transfer agent having a transition metal complex, and analyte sensors with enzyme layers that include immobilized NAD(P)+ or derivative thereof and an electron transfer agent comprising a transition metal complex. Embodiments of the present disclosure relate to enzyme compositions for analyte sensing including where the subject compositions provide for monitoring of an analyte in vivo over an extended period of time. Where the subject enzyme compositions include an NAD(P)+-dependent dehydrogenase, analyte sensors described herein provide for clinically accurate electrochemical measurement of analytes that are catalyzed by an NAD(P)+-dependent dehydrogenase. As described in greater detail below, the subject enzyme compositions provide for clinically accurate electrochemical measurement of analytes as measured by Clark error grid analysis and/or MARD analysis and/or MAD analysis. In particular, the subject enzyme compositions provide for measurement by an analyte sensor incorporating the subject compositions to produce a signal that increases linearly as a function of analyte concentration. In addition, the subject enzyme compositions provide for clinically accurate electrochemical measurement of analytes that are catalyzed by an NAD(P)+-dependent dehydrogenase within 30 seconds of contacting a fluidic sample (e.g., interstitial fluid when the sensor is positioned beneath the surface of a subject&#39;s skin) with the sensor. In certain instances, the subject enzyme compositions provide for clinically accurate electrochemical measurements of analytes that are catalyzed by an NAD(P)+-dependent dehydrogenase immediately after contacting the fluidic sample with the sensor. 
     The subject enzyme compositions include an NAD(P)+-dependent dehydrogenase, such as glucose dehydrogenase, alcohol dehydrogenase or D-3-hydroxybutyrate dehydrogenase. In some embodiments, NAD(P)+-dependent dehydrogenases of interest are oxidoreductases belonging to the enzyme class 1.1.1- 
     The NAD(P)+-dependent dehydrogenase may be present in the subject compositions in an amount that varies, such as from 0.05 μg to 5 μg, such as from 0.1 μg to 4 μg, such as from 0.2 μg to 3 μg and including from 0.5 μg to 2 μg. As such, the amount of NAD(P)+-dependent dehydrogenase is from 0.01% to 10% by weight of the total enzyme composition, such as from 0.05% to 9.5% by weight, such as from 0.1% to 9% by weight, such as 0.5% to 8.5% by weight, such as from 1% to 8% by weight and including from 2% to 7% by weight of the total enzyme composition. 
     Enzyme compositions also include nicotinamide adenine dinucleotide phosphate (NAD(P)+) or derivative thereof In some embodiments, enzyme compositions of interest include nicotinamide adenine dinucleotide phosphate (NAD(P)+). In other embodiments, enzyme compositions include a derivative of nicotinamide adenine dinucleotide phosphate (NAD(P)+). Derivatives of nicotinamide adenine dinucleotide phosphate (NAD(P)+) may include compounds of Formula I: 
     
       
         
         
             
             
         
       
     
     where X is alkyl, substituted alkyl, aryl, substituted aryl, acyl, and aminoacyl. 
     In some embodiments, X is an aminoacyl substituted alkyl. In some embodiments, X is CH2C(O)NH(CH2) y NH2 where y is an integer from 1 to 10, such as 2 to 9, such as 3 to 8 and including where y is 6. In certain instances, X is CH2C(O)NH(CH2)6NH2. In these embodiments, the derivative of nicotinamide adenine dinucleotide phosphate (NAD(P) + ) in the subject enzyme composition is: 
     
       
         
         
             
             
         
       
     
     Embodiments of the enzyme composition also include an NAD(P)H oxidoreductase. In certain embodiments, the enzyme composition includes diaphorase The amount of NAD(P)H oxidoreductase (e.g., diaphorase) present in the subject compositions ranges from 0.01 μg to 10 μg, such as from 0.02 μg to 9 μg, such as from 0.03 μg to 8 μg, such as from 0.04 μg to 7 μg, such as from 0.05 μg to 5 μg, such as from 0.1 μg to 4 μg, such as from 0.2 μg to 3 μg and including from 0.5 μg to 2 μg. As such, the amount of NAD(P)H oxidoreductase (e.g., diaphorase) is from 0.01% to 10% by weight of the total enzyme composition, such as from 0.05% to 9.5% by weight, such as from 0.1% to 9% by weight, such as 0.5% to 8.5% by weight, such as from 1% to 8% by weight and including from 2% to 7% by weight of the total enzyme composition. 
     In some embodiments, the weight ratio of NAD(P)+-dependent dehydrogenase to NAD(P)H oxidoreductase (e.g., diaphorase) ranges from 1 to 10 NAD(P)+-dependent dehydrogenase to NAD(P)H oxidoreductase, such as from 1 to 8, such as from 1 to 5, such as from 1 to 2 and including from 1 to 1 NAD(P)+-dependent dehydrogenase to NAD(P)H oxidoreductase. In other embodiments, the weight ratio of NAD(P)+-dependent dehydrogenase to NAD(P)H oxidoreductase ranges from 10 to 1 NAD(P)+-dependent dehydrogenase to NAD(P)H oxidoreductase, such as from 8 to 1, such as from 5 to 1 and including from 2 to 1 NAD(P)+-dependent dehydrogenase to NAD(P)H oxidoreductase. 
     Enzyme compositions of interest also include an electron transfer agent having an transition metal complex. They may be electroreducible and electrooxidizable ions or molecules having redox potentials that are a few hundred millivolts above or below the redox potential of the standard calomel electrode (SCE). Examples of transition metal complexes include metallocenes including ferrocene, hexacyanoferrate (III), ruthenium hexamine, etc. Additional examples include those described in U.S. Pat. Nos. 6,736,957, 7,501,053 and 7,754,093, the disclosures of each of which are incorporated herein by reference in their entirety. 
     In some embodiments, electron transfer agents are osmium transition metal complexes with one or more ligands, each ligand having a nitrogen-containing heterocycle such as 2,2′-bipyridine, 1,10-phenanthroline, 1-methyl, 2-pyridyl biimidazole, or derivatives thereof. The electron transfer agents may also have one or more ligands covalently bound in a polymer, each ligand having at least one nitrogen-containing heterocycle, such as pyridine, imidazole, or derivatives thereof. One example of an electron transfer agent includes (a) a polymer or copolymer having pyridine or imidazole functional groups and (b) osmium cations complexed with two ligands, each ligand containing 2,2′-bipyridine, 1,10-phenanthroline, or derivatives thereof, the two ligands not necessarily being the same. Some derivatives of 2,2′-bipyridine for complexation with the osmium cation include but are not limited to 4,4′-dimethyl-2,2′-bipyridine and mono-, di-, and polyalkoxy-2,2′-bipyridines, including 4,4′-dimethoxy-2,2′-bipyridine. Derivatives of 1,10-phenanthroline for complexation with the osmium cation include but are not limited to 4,7-dimethyl-1,10-phenanthroline and mono, di-, and polyalkoxy-1,10-phenanthrolines, such as 4,7-dimethoxy-1,10-phenanthroline. Polymers for complexation with the osmium cation include but are not limited to polymers and copolymers of poly(1-vinyl imidazole) (referred to as “PVI”) and poly(4-vinyl pyridine) (referred to as “PVP”). Suitable copolymer substituents of poly(1-vinyl imidazole) include acrylonitrile, acrylamide, and substituted or quaternized N-vinyl imidazole, e.g., electron transfer agents with osmium complexed to a polymer or copolymer of poly(1-vinyl imidazole). 
     The subject enzyme compositions may be heterogeneous or homogenous. In some embodiments, each component (i.e., nicotinamide adenine dinucleotide phosphate (NAD(P)+) or derivative thereof, an NAD(P)+-dependent dehydrogenase, an NAD(P)H oxidoreductase and an electron transfer agent having a transition metal complex) is uniformly distributed throughout the composition, e.g., when applied to an electrode, as described in greater detail below. For example, each of nicotinamide adenine dinucleotide phosphate (NAD(P)+) or derivative thereof, an NAD(P)+-dependent dehydrogenase, an NAD(P)H oxidoreductase and an electron transfer agent having a transition metal complex may be distributed uniformly throughout the composition, such that the concentration of each component is the same throughout. 
     In certain embodiments, the subject enzyme compositions described herein are polymeric. Polymers that may be used may be branched or unbranched and may be homopolymers formed from the polymerization of a single type of monomer or heteropolymers that include two or more different types of monomers. Heteropolymers may be copolymers where the copolymer has alternating monomer subunits, or in some cases, may be block copolymers, which include two or more homopolymer subunits linked by covalent bonds (e.g, diblock or triblock copolymers). In some embodiments, the subject enzyme compositions include a heterocycle-containing polymer. The term heterocycle (also referred to as “heterocycicyl”) is used herein in its conventional sense to refer to any cyclic moiety which includes one or more heteroatoms (i.e., atoms other than carbon) and may include, but are not limited to N, P, O, S, Si, etc. Heterocycle-containing polymers may be heteroalkyl, heteroalkanyl, heteroalkenyl and heteroalkynyl as well as heteroaryl or heteroarylalkyl. 
     “Heteroalkyl, Heteroalkanyl, Heteroalkenyl and Heteroalkynyl” by themselves or as part of another substituent refer to alkyl, alkanyl, alkenyl and alkynyl groups, respectively, in which one or more of the carbon atoms (and any associated hydrogen atoms) are independently replaced with the same or different heteroatomic groups. Typical heteroatomic groups which can be included in these groups include, but are not limited to, —O—, —S—, —S—S—, —O—S—, —NR37R38-, ═N—N═, —N═N—, —N═N—NR39R40, —PR41—, —P(O)2-, —POR42-, —O—P(O)2—, —S—O—, —S—(O)—, —SO2—, —SnR43R44- and the like, where R37, R38, R39, R40, R41, R42, R43 and R44 are independently hydrogen, alkyl, substituted alkyl, aryl, substituted aryl, arylalkyl, substituted arylalkyl, cycloalkyl, substituted cycloalkyl, cycloheteroalkyl, substituted cycloheteroalkyl, heteroalkyl, substituted heteroalkyl, heteroaryl, substituted heteroaryl, heteroarylalkyl or substituted heteroarylalkyl. 
     “Heteroaryl” by itself or as part of another substituent, refers to a monovalent heteroaromatic radical derived by the removal of one hydrogen atom from a single atom of a heteroaromatic ring system. Typical heteroaryl groups include, but are not limited to, groups derived from acridine, arsindole, carbazole, β-carboline, chromane, chromene, cinnoline, furan, imidazole, indazole, indole, indoline, indolizine, isobenzofuran, isochromene, isoindole, isoindoline, isoquinoline, isothiazole, isoxazole, naphthyridine, oxadiazole, oxazole, perimidine, phenanthridine, phenanthroline, phenazine, phthalazine, pteridine, purine, pyran, pyrazine, pyrazole, pyridazine, pyridine, pyrimidine, pyrrole, pyrrolizine, quinazoline, quinoline, quinolizine, quinoxaline, tetrazole, thiadiazole, thiazole, thiophene, triazole, xanthene, benzodioxole and the like. In certain embodiments, the heteroaryl group is from 5-20 membered heteroaryl. In certain embodiments, the heteroaryl group is from 5-10 membered heteroaryl. In certain embodiments, heteroaryl groups are those derived from thiophene, pyrrole, benzothiophene, benzofuran, indole, pyridine, quinoline, imidazole, oxazole and pyrazine. 
     “Heteroarylalkyl” by itself or as part of another substituent, refers to an acyclic alkyl radical in which one of the hydrogen atoms bonded to a carbon atom, typically a terminal or sp3 carbon atom, is replaced with a heteroaryl group. Where specific alkyl moieties are intended, the nomenclature heteroarylalkanyl, heteroarylalkenyl and/or heterorylalkynyl is used. In certain embodiments, the heteroarylalkyl group is a 6-30 membered heteroarylalkyl, e.g., the alkanyl, alkenyl or alkynyl moiety of the heteroarylalkyl is 1-10 membered and the heteroaryl moiety is a 5-20-membered heteroaryl. In certain embodiments, the heteroarylalkyl group is 6-20 membered heteroarylalkyl, e.g., the alkanyl, alkenyl or alkynyl moiety of the heteroarylalkyl is 1-8 membered and the heteroaryl moiety is a 5-12-membered heteroaryl. 
     In some embodiments, the heterocycle-containing component is an aromatic ring system. “Aromatic Ring System” by itself or as part of another substituent, refers to an unsaturated cyclic or polycyclic ring system having a conjugated π electron system. Specifically included within the definition of “aromatic ring system” are fused ring systems in which one or more of the rings are aromatic and one or more of the rings are saturated or unsaturated, such as, for example, fluorene, indane, indene, phenalene, etc. Typical aromatic ring systems include, but are not limited to, aceanthrylene, acenaphthylene, acephenanthrylene, anthracene, azulene, benzene, chrysene, coronene, fluoranthene, fluorene, hexacene, hexaphene, hexalene, as-indacene, s-indacene, indane, indene, naphthalene, octacene, octaphene, octalene, ovalene, penta-2,4-diene, pentacene, pentalene, pentaphene, perylene, phenalene, phenanthrene, picene, pleiadene, pyrene, pyranthrene, rubicene, triphenylene, trinaphthalene and the like. 
     “Heteroaromatic Ring System” by itself or as part of another substituent, refers to an aromatic ring system in which one or more carbon atoms (and any associated hydrogen atoms) are independently replaced with the same or different heteroatom. Typical heteroatoms to replace the carbon atoms include, but are not limited to, N, P, O, S, Si, etc. Specifically included within the definition of “heteroaromatic ring systems” are fused ring systems in which one or more of the rings are aromatic and one or more of the rings are saturated or unsaturated, such as, for example, arsindole, benzodioxan, benzofuran, chromane, chromene, indole, indoline, xanthene, etc. Typical heteroaromatic ring systems include, but are not limited to, arsindole, carbazole, β-carboline, chromane, chromene, cinnoline, furan, imidazole, indazole, indole, indoline, indolizine, isobenzofuran, isochromene, isoindole, isoindoline, isoquinoline, isothiazole, isoxazole, naphthyridine, oxadiazole, oxazole, perimidine, phenanthridine, phenanthroline, phenazine, phthalazine, pteridine, purine, pyran, pyrazine, pyrazole, pyridazine, pyridine, pyrimidine, pyrrole, pyrrolizine, quinazoline, quinoline, quinolizine, quinoxaline, tetrazole, thiadiazole, thiazole, thiophene, triazole, xanthene and the like. 
     In certain embodiments, enzyme compositions of interest include a heterocyclic nitrogen containing component, such as polymers of polyvinylpyridine (PVP) and polyvinylimidazole. 
     The polymeric enzyme compositions may also include one or more crosslinkers (crosslinking agent) such that the polymeric backbone enzyme composition is crosslinked. As described herein, reference to linking two or more different polymers together is intermolecular crosslinking, whereas linking two more portions of the same polymer is intramolecular crosslinking. In embodiments of the present disclosure, crosslinkers may be capable of both intermolecular and intramolecular crosslinkings at the same time. 
     Suitable crosslinkers may be bifunctional, trifunctional or tetrafunctional, each having straight chain or branched structures. Crosslinkers having branched structures include a multi-arm branching component, such as a 3-arm branching component, a 4-arm branching component, a 5-arm branching component, a 6-arm branching component or a larger number arm branching component, such as having 7 arms or more, such as 8 arms or more, such as 9 arms or more, such as 10 arms or more and including 15 arms or more. In certain instances, the multi-arm branching component is a multi-arm epoxide, such as 3-arm epoxide or a 4-arm epoxide. Where the multi-arm branching component is a multi-arm epoxide, the multi-arm branching component may be a polyethylene glycol (PEG) multi-arm epoxide or a non-polyethylene glycol (non-PEG) multi-arm epoxide. In some embodiments, the multi-arm branching component is a non-PEG multi-arm epoxide. In other embodiments, the multi-arm branching component is a PEG multi-arm epoxide. In certain embodiments, the multi-arm branching component is a 3-arm PEG epoxide or a 4-arm PEG epoxide. 
     Examples of crosslinkers include but are not limited to polyethylene glycol diglycidyl ether, N,N-diglycidyl-4-glycidyloxyaniline as well as nitrogen-containing multifunctional crosslinkers having the structures: 
     
       
         
         
             
             
         
       
     
     In some instances, one or more bonds with the one or more components of the enzyme composition may be formed such as between one or more of the nicotinamide adenine dinucleotide phosphate (NAD(P)+) or derivative thereof, NAD(P)+-dependent dehydrogenase, NAD(P)H oxidoreductase and electron transfer agent. By bonds is meant any type of an interaction between atoms or molecules that allows chemical compounds to form associations with each other, such as, but not limited to, covalent bonds, ionic bonds, dipole-dipole interactions, hydrogen bonds, London dispersion forces, and the like. For example, in situ polymerization of the enzyme compositions can form crosslinks between the polymers of the composition and the NAD(P)+-dependent dehydrogenase, nicotinamide adenine dinucleotide phosphate (NAD(P)+) or derivative thereof, the NAD(P)H oxidoreductase and the electron transfer agent. In certain embodiments, crosslinking of the polymer to the one or more of the NAD(P)+-dependent dehydrogenase, nicotinamide adenine dinucleotide phosphate (NAD(P)+) or derivative thereof, the NAD(P)H oxidoreductase and the electron transfer agent facilitates a reduction in the occurrence of delamination of the enzyme compositions from an electrode. 
     As described herein, the subject enzyme may be used in an analyte sensor to monitor the concentration of an NAD(P)+-dependent dehydrogenase analyte, such as glucose, an alcohol, a ketone, lactate, or β-hydroxybutyrate, and the sensor may have one or more electrodes with the enzyme composition. In embodiments, the analyte sensor includes: a working electrode having a conductive material the subject enzyme composition proximate to (e.g., disposed on) and in contact with the conductive material. One or more other electrode may be included such as one or more counter electrodes, one or more reference electrodes and/or one or more counter/reference electrodes. 
     The particular configuration of electrochemical sensors may depend on the use for which the analyte sensor is intended and the conditions under which the analyte sensor will operate. In certain embodiments of the present disclosure, analyte sensors are in vivo wholly positioned analyte sensors or transcutaneously positioned analyte sensors configured for in vivo positioning in a subj ect. In one example, at least a portion of the sensor may be positioned in the subcutaneous tissue for testing lactate concentrations in interstitial fluid. In another example, at least a portion of the sensor may be positioned in the dermal tissue for testing analyte concentration in dermal fluid. 
     In embodiments, one or more of the subject enzyme compositions is positioned proximate to (e.g., disposed on) the surface of a working electrode. In some instances, a plurality of enzyme compositions are positioned proximate to the surface of working electrode (e.g., in the form of spots). In certain cases, a discontinuous or continuous perimeter is formed around each of the plurality of enzyme compositions positioned proximate to the surface of the working electrode. Examples of depositing a plurality of reagent compositions to the surface of an electrode as well as forming a discontinuous or continuous perimeter around each reagent composition is described in U.S. Patent Publication No. 2012/0150005 and in co-pending U.S. Patent Application No. 62/067,813, the disclosures of which are herein incorporated by reference. 
     The subject enzyme compositions having nicotinamide adenine dinucleotide phosphate (NAD(P)+) or derivative thereof, NAD(P)+-dependent dehydrogenase, NAD(P)H oxidoreductase and electron transfer agent may be deposited onto the surface of the working electrode as one large application which covers the desired portion of the working electrode or in the form of an array of a plurality of enzyme compositions, e.g., spaced apart from each other. Depending upon use, any or all of the enzyme compositions in the array may be the same or different from one another. For example, an array may include two or more, 5 or more enzyme composition array features containing nicotinamide adenine dinucleotide phosphate (NAD(P)+) or derivative thereof, NAD(P)+-dependent dehydrogenase, NAD(P)H oxidoreductase and electron transfer agent, 10 or more, 25 or more, 50 or more, 100 or more, or even 1000 or more, in an area of 100 mm 2  or less, such as 75 mm 2  or less, or 50 mm 2  or less, for instance 25 mm 2  or less, or 10 mm 2  or less, or 5 mm 2  or less, such as 2 mm 2  or less, or 1 mm 2  or less, 0.5 mm 2  or less, or 0.1 mm 2  or less. 
     The shape of deposited enzyme composition may vary within or between sensors. For example, in certain embodiments, the deposited membrane is circular. In other embodiments, the shape will be of a triangle, square, rectangle, circle, ellipse, or other regular or irregular polygonal shape (e.g., when viewed from above) as well as other two-dimensional shapes such as a circle, half circle or crescent shape. All or a portion of the electrode may be covered by the enzyme composition, such as 5% or more, such as 25% or more, such as 50% or more, such as 75% or more and including 90% or more. In certain instances, the entire electrode surface is covered by the enzyme composition (i.e., 100%). 
     Fabricating an electrode and/or sensor according to embodiments of the present disclosure produces a reproducible enzyme composition deposited on the surface of the electrode. For example, enzyme compositions provided herein may deviate from each other by 5% or less, such as by 4% or less, such as by 3% or less, such as by 2% or less, such as by 1% or less and including by 0.5% or less. In some embodiments, the sensing composition includes nicotinamide adenine dinucleotide phosphate (NAD(P)+) or derivative thereof and an electron transfer agent. In certain embodiments, deposited enzyme compositions containing nicotinamide adenine dinucleotide phosphate (NAD(P)+) or derivative thereof, NAD(P)+-dependent dehydrogenase, NAD(P)H oxidoreductase and electron transfer agent show no deviation from one another and are identical. 
     In certain embodiments, methods further include drying the enzyme composition deposited on the electrode. Drying may be performed at room temperature, at an elevated temperature, as desired, such as at a temperature ranging from 25° C. to 100° C., such as from 30° C. to 80° C. and including from 40° C. to 60° C. 
     Examples of configurations for the subject analyte sensors and methods for fabricating them may include, but are not limited to, those described in U.S. Pat. Nos. 6,175,752, 6,134,461, 6,579,690, 6,605,200, 6,605,201, 6,654,625, 6,746,582, 6,932,894, 7,090,756, 5,356,786, 6,560,471, 5,262,035, 6,881,551, 6,121,009, 6,071,391, 6,377,894, 6,600,997, 6,514,460, 5,820,551, 6,736,957, 6,503,381, 6,676,816, 6,514,718, 5,593,852, 6,284,478, 7,299,082, 7,811,231, 7,822,557 8,106,780, and 8,435,682; U.S. Patent Application Publication Nos. 2010/0198034, 2010/0324392, 2010/0326842, 2007/0095661, 2010/0213057, 2011/0120865, 2011/0124994, 2011/0124993, 2010/0213057, 2011/0213225, 2011/0126188, 2011/0256024, 2011/0257495, 2012/0157801, 2012/0245447, 2012/0157801, 2012/0323098, and 20130116524, the disclosures of each of which are incorporated herein by reference in their entirety. 
     In some embodiments, in vivo sensors may include an insertion tip positionable below the surface of the skin, e.g., penetrating through the skin and into, e.g., the subcutaneous space, in contact with the user&#39;s biological fluid such as interstitial fluid. Contact portions of working electrode, a reference electrode and a counter electrode are positioned on the first portion of the sensor situated above the skin surface. A working electrode, a reference electrode and a counter electrode are positioned at the inserted portion of the sensor. Traces may be provided from the electrodes at the tip to a contact configured for connection with sensor electronics. 
     In certain embodiments, the working electrode and counter electrode of the sensor as well as dielectric material of are layered. For example, the sensor may include a non-conductive material layer, and a first conductive layer such as conductive polymer, carbon, platinum-carbon, gold, etc., disposed on at least a portion of the non-conductive material layer (as described above). The enzyme composition is positioned on one or more surfaces of the working electrode, or may otherwise be directly or indirectly contacted to the working electrode. A first insulation layer, such as a first dielectric layer may disposed or layered on at least a portion of a first conductive layer and a second conductive layer may be positioned or stacked on top of at least a portion of a first insulation layer (or dielectric layer). The second conductive layer may be a reference electrode. A second insulation layer, such as a second dielectric layer may be positioned or layered on at least a portion of the second conductive layer. Further, a third conductive layer may be positioned on at least a portion of the second insulation layer and may be a counter electrode. Finally, a third insulation layer may be disposed or layered on at least a portion of the third conductive layer. In this manner, the sensor may be layered such that at least a portion of each of the conductive layers is separated by a respective insulation layer (for example, a dielectric layer). 
     In other embodiments, some or all of the electrodes may be provided in a co-planar manner such that two or more electrodes may be positioned on the same plane (e.g., side-by side (e.g., parallel) or angled relative to each other) on the material. For example, co-planar electrodes may include a suitable spacing there between and/or include a dielectric material or insulation material disposed between the conductive layers/electrodes. Furthermore, in certain embodiments one or more of the electrodes may be disposed on opposing sides of the nonconductive material. In such embodiments, electrical contact may be on the same or different sides of the non-conductive material. For example, an electrode may be on a first side and its respective contact may be on a second side, e.g., a trace connecting the electrode and the contact may traverse through the material. A via provides an avenue through which an electrical trace is brought to an opposing side of a sensor. 
     The subject analyte sensors be configured for monitoring the level of an analyte (e.g., glucose, an alcohol, a ketone, lacate, β-hydroxybutyrate) over a time period which may range from seconds, minutes, hours, days, weeks, to months, or longer. 
     In certain embodiments, the analyte sensor includes a mass transport limiting layer (or a membrane layer), e.g., an analyte flux modulating layer, to act as a diffusion-limiting barrier to reduce the rate of mass transport of the analyte, for example, glucose, an alcohol, a ketone, lactate, β-hydroxybutyrate, when the sensor is in use. The mass transport limiting layers limit the flux of an analyte to the electrode in an electrochemical sensor so that the sensor is linearly responsive over a large range of analyte concentrations. Mass transport limiting layers may include polymers and may be biocompatible. A mass transport limiting layer may provide many functions, e.g., biocompatibility and/or interferent-eliminating functions, etc., or functions may be provided by various membrane layers. 
     In certain embodiments, a mass transport limiting layer is a membrane composed of crosslinked polymers containing heterocyclic nitrogen groups, such as polymers of polyvinylpyridine and polyvinylimidazole. Embodiments also include membranes that are made of a polyurethane, or polyether urethane, or chemically related material, or membranes that are made of silicone, and the like. 
     The membrane may be formed by crosslinking in situ a polymer, modified with a zwitterionic moiety, a non-pyridine copolymer component, and optionally another moiety that is either hydrophilic or hydrophobic, and/or has other desirable properties, in an alcohol-buffer solution. The modified polymer may be made from a precursor polymer containing heterocyclic nitrogen groups. For example, a precursor polymer may be polyvinylpyridine or polyvinylimidazole. Optionally, hydrophilic or hydrophobic modifiers may be used to “fine-tune” the permeability of the resulting membrane to an analyte of interest. Optional hydrophilic modifiers, such as poly(ethylene glycol), hydroxyl or polyhydroxyl modifiers, may be used to enhance the biocompatibility of the polymer or the resulting membrane. 
     Suitable mass transport limiting membranes in the subject analyte sensors may include, but are not limited to those described in U.S. Pat. No. 6,932,894, the disclosure of which is herein incorporated by reference. In certain embodiments, the mass transport limiting membrane is a SMART membrane that is temperature independent. Suitable temperature independent membranes may include, but are not limited to those described in U.S. Patent Publication No. 2012/0296186 and copending U.S. patent application Ser. No. 14/737,082, the disclosures of which are herein incorporated by reference. 
     Analyte sensors according to certain embodiments may be configured to operate at low oxygen concentration. By low oxygen concentration is meant the concentration of oxygen is 1.5 mg/L or less, such as 1.0 mg/L or less, such as 0.75 mg/L or less, such as 0.6 mg/L or less, such as 0.3 mg/L or less, such as 0.25 mg/L or less, such as 0.15 mg/L or less, such as 0.1 mg/L or less and including 0.05 mg/L or less. 
     Aspects of the present disclosure also include methods for in vivo monitoring analyte levels over time with analyte sensors incorporating an enzyme composition containing nicotinamide adenine dinucleotide phosphate (NAD(P)+) or derivative thereof, NAD(P)+-dependent dehydrogenase, NAD(P)H oxidoreductase and electron transfer agent. Generally, in vivo monitoring the concentration of analyte in a fluid of the body of a subject includes inserting at least partially under a skin surface an in vivo analyte sensor as disclosed herein, contacting the monitored fluid (interstitial, blood, dermal, and the like) with the inserted sensor, and generating a sensor signal at the working electrode. The presence and/or concentration of analyte detected by the analyte sensor may be displayed, stored, forwarded, and/or otherwise processed. A variety of approaches may be employed to determine the concentration of analyte (e.g., glucose, an alcohol, a ketone, lacate, β-hydroxybutyrate) with the subject sensors. In certain aspects, an electrochemical analyte concentration monitoring approach is used. For example, monitoring the concentration of analyte using the sensor signal may be performed by coulometric, amperometric, voltammetric, potentiometric, or any other convenient electrochemical detection technique. 
     These methods may also be used in connection with a device that is used to detect and/or measure another analyte, including glucose, oxygen, carbon dioxide, electrolytes, or other moieties of interest, for example, or any combination thereof, found in a bodily fluid, including subcutaneous e.g., interstitial fluid, dermal fluid, blood or other bodily fluid of interest or any combination thereof. 
     In certain embodiments, the method further includes attaching an electronics unit to the skin of the patient, coupling conductive contacts of the electronics unit to contacts of the sensor, collecting data using the electronics unit regarding a level of analyte from signals generated by the sensor, and forwarding the collected data from electronics unit to a receiver unit, e.g., by RF. The receiver unit may be a mobile telephone. The mobile telephone may include a application related to the monitored analyte. In certain embodiments, analyte information is forwarded by RFID protocol, Bluetooth, and the like. 
     The analyte sensor may be positionable in a user for automatic analyte sensing, either continuously or periodically. Embodiments may include monitoring the level of the analyte over a time period which may range from seconds, minutes, hours, days, weeks, to months, or longer. Future analyte levels may be predicted based on information obtained, e.g., the current lactate level at time zero as well as an analyte rate of change. 
     The sensor electronics unit may automatically forward data from the sensor/electronics unit to one or more receiver units. The sensor data may be communicated automatically and periodically, such as at a certain frequency as data is obtained or after a certain time period of sensor data stored in memory. For example, sensor electronics coupled to an in vivo positioned sensor may collect the sensor data for a predetermined period of time and transmit the collected data periodically (e.g., every minute, five minutes, or other predetermined period) to a monitoring device that is positioned in range from the sensor electronics. 
     In other embodiments, the sensor electronics coupled to the in vivo positioned sensor may communicate with the receiving device non automatically manner and not set to any specific schedule. For example, the sensor data may be communicated from the sensor electronics to the receiving device using RFID technology, and communicated whenever the sensor electronics are brought into communication range of the analyte monitoring device. For example, the in vivo positioned sensor may collect sensor data in memory until the monitoring device (e.g., receiver unit) is brought into communication range of the sensor electronics unit—e.g., by the patient or user. When the in vivo positioned sensor is detected by the monitoring device, the device establishes communication with the analyte sensor electronics and uploads the sensor data that has been collected since the last transfer of sensor data, for instance. In this way, the patient does not have to maintain close proximity to the receiving device at all times, and instead, can upload sensor data when desired by bringing the receiving device into range of the analyte sensor. In yet other embodiments, a combination of automatic and non-automatic transfers of sensor data may be implemented in certain embodiments. For example, transfers of sensor data may be initiated when brought into communication range, and then continued on an automatic basis if continued to remain in communication range. 
     Example Embodiments of Calibration 
     Biochemical sensors can be described by one or more sensing characteristics. A common sensing characteristic is referred to as the biochemical sensor&#39;s sensitivity, which is a measure of the sensor&#39;s responsiveness to the concentration of the chemical or composition it is designed to detect. For electrochemical sensors, this response can be in the form of an electrical current (amperometric) or electrical charge (coulometric). For other types of sensors, the response can be in a different form, such as a photonic intensity (e.g., optical light). The sensitivity of a biochemical analyte sensor can vary depending on a number of factors, including whether the sensor is in an in vitro state or an in vivo state. 
       FIG. 15  is a graph depicting the in vitro sensitivity of an amperometric analyte sensor. The in vitro sensitivity can be obtained by in vitro testing the sensor at various analyte concentrations and then performing a regression (e.g., linear or non-linear) or other curve fitting on the resulting data. In this example, the analyte sensor&#39;s sensitivity is linear, or substantially linear, and can be modeled according to the equation y=mx+b, where y is the sensor&#39;s electrical output current, x is the analyte level (or concentration), m is the slope of the sensitivity and b is the intercept of the sensitivity, where the intercept generally corresponds to a background signal (e.g., noise). For sensors with a linear or substantially linear response, the analyte level that corresponds to a given current can be determined from the slope and intercept of the sensitivity. Sensors with a non-linear sensitivity require additional information to determine the analyte level resulting from the sensor&#39;s output current, and those of ordinary skill in the art are familiar with manners by which to model non-linear sensitivities. In certain embodiments of in vivo sensors, the in vitro sensitivity may be the same as the in vivo sensitivity, but in other embodiments a transfer (or conversion) function is used to translate the in vitro sensitivity into the in vivo sensitivity that is applicable to the sensor&#39;s intended in vivo use. 
     Examples of Sensing Characteristics Derived from Testing 
     As described, one or more medical devices within the baseline subset can be tested to empirically determine a sensing characteristic for that baseline subset. The testing is, in many embodiments, capable of producing data that verifiably represents the ability of the medical device to sense the biochemical attribute. In many in vivo analyte sensor and in vitro analyte sensor (e.g., test strip) embodiments, the sensing characteristic can be the sensitivity of the analyte sensor to the presence of the analyte. Often this testing will be performed in vitro and will result in the collection of in vitro test data. The sensing characteristic derived or otherwise resulting from the in vitro test data for the baseline subset can be referred to as an in vitro sensing characteristic (e.g., in vitro sensitivity). 
     The following examples are put forth so as to provide those of ordinary skill in the art with a complete disclosure and description of how to make and use the embodiments of the invention, and are not intended to limit the scope of what the inventors regard as their invention nor are they intended to represent that the experiments below are all or the only experiments performed. Efforts have been made to ensure accuracy with respect to numbers used (e.g., amounts, temperature, etc.) but some experimental errors and deviations should be accounted for. Unless indicated otherwise, parts are parts by weight, molecular weight is weight average molecular weight, temperature is in degrees Centigrade, and pressure is at or near atmospheric. 
     EXAMPLE 1 
     Experiments were performed to demonstrate the performance of analyte sensors having a working electrode that contains nicotinamide adenine dinucleotide phosphate (NAD(P) + ) or derivative thereof, NAD(P) + -dependent dehydrogenase, NAD(P)H oxidoreductase and electron transfer agent. The sensors were prepared by depositing onto the surface of an electrode an enzyme composition containing nicotinamide adenine dinucleotide phosphate, D-3-hydroxybutyrate dehydrogenase, diaphorase and a polymer bound osmium-transition metal catalyst and a difunctional crosslinker, as shown by the scheme (also referred to as a polymeric redox mediator in further examples): 
     
       
         
         
             
             
         
       
     
     The sensors were tested in phosphate buffer containing varying concentrations of D-3-hydroxybutyrate. Table 1 summarizes beaker calibration and linearity of data signal from the prepared sensors. 
     
       
         
           
               
               
             
               
                   
                 TABLE 1 
               
               
                   
                   
               
               
                   
                 D-3-Hydroxybutyrate Sensor 
               
               
                   
                   
               
             
            
               
                   
               
            
           
           
               
               
               
            
               
                   
                 Slope 
                 0.0163 
               
               
                   
                 R2 
                 0.9982 
               
               
                   
                   
               
            
           
         
       
     
       FIG. 16  shows the signal output over the course of 2.3 hours at varying concentrations of D-3-hydroxybutyrate (80 μM, 160 μM and 240 μM).  FIG. 17  depicts the linearity of the sensor signal as a function of D-3-hydroxybutyrate concentration. As shown in  FIGS. 1 and 2 , the sensor gives a linear and persistent response to D-3-hydroxybutyrate. 
     Additional enzymes for detecting kentoes are described in PCT Application No. PCT/US21/62968, U.S. Pat. No. 11,091,788, and U.S. Patent Application No. 2020/0237275 , the disclosure of which is incorporated by reference in its entirety. 
     EXAMPLE 2 
     Experiments were performed to demonstrate the performance of analyte sensors having a working electrode that contains free NAD. The sensor was prepared by depositing onto the surface of an electrode an enzyme composition containing the free NAD. Sensing layer formulation is described in Table 2. The sensing layer solutions was deposit on carbon electrodes, and cured at 25C/60H overnight, prior to addition of membrane. The membrane formulation is provided in Table 3. The sensor was dipped from above solution 3×5 mm/sec, and the sensor was cured at 25C/60H overnight, 56C for two days. 
     
       
         
           
               
             
               
                 TABLE 2 
               
             
            
               
                   
               
               
                 Formulation Table 
               
               
                 Sensing Layer Solution 
               
               
                 BD161212 
               
            
           
           
               
               
               
               
               
               
            
               
                   
                   
                   
                   
                 Mixing 
                 Final 
               
               
                 10 mM Hepes 
                 Vendor 
                 Cat 
                 mg/mL 
                 uL 
                 mg/mL 
               
               
                   
               
            
           
           
               
               
               
               
               
               
            
               
                 D-3-Hydroxybutyrate 
                 Toyobo 
                 HBD-301 
                 40 
                 20 
                 8.89 
               
               
                 Dehydrogenase (HBD) 
               
               
                 Diaphorase 
                 Toyobo 
                 DAD-311 
                 40 
                 20 
                 8.89 
               
               
                 NAD 
                 Sigma 
                 N0632 
                 20 
                 10 
                 2.22 
               
               
                 Polymeric redox 
                   
                   
                 40 
                 20 
                 8.89 
               
               
                 mediator 
               
               
                 Peg400 
                   
                   
                 40 
                 20 
                 8.89 
               
               
                 Total 
                   
                   
                   
                 90 
               
               
                   
               
            
           
         
       
     
     
       
         
           
               
             
               
                 TABLE 3 
               
               
                   
               
               
                 Membrane Coating 
               
               
                 Membrane Solution 
               
               
                   
               
             
            
               
                   
               
            
           
           
               
               
               
               
               
               
            
               
                 Solution A 
                 Vendor 
                 Cat 
                 MW 
                 Mixing 
               
               
                   
               
               
                 Poly(4-vinylpyridine) 
                 Sigma 
                 472352 
                 160K 
                 120 
                 mg 
               
               
                 Ethanol/Hepe (10 mM pH 8.0) 
                   
                   
                   
                 1 
                 mL 
               
               
                   
               
            
           
           
               
               
               
               
               
            
               
                 Solution B 
                 Vendor 
                 Cat 
                 Mixing 
               
               
                   
               
               
                 Poly(ethylene glycol) diglycidyl 
                 Polysciences 
                 8210 
                 100 
                 mg 
               
               
                 ether (PEGDGE 400) 
               
               
                 Ethanol/Hepe (10 mM pH 8.0) 
                   
                   
                 1 
                 mL 
               
               
                   
               
            
           
           
               
               
               
            
               
                   
                 Final membrane solution 
                 Mixing (mL) 
               
               
                   
                   
               
               
                   
                 Solution A 
                 4 
               
               
                   
                 Solution B 
                 0.4 
               
               
                   
                   
               
            
           
         
       
     
       FIG. 18  shows the signal output over the course of 3.6 hours at varying concentrations of D-3-hydroxybutyrate (ketone).  FIG. 19  depicts the linearity of the sensor signal as a function of D-3-hydroxybutyrate concentration.  FIG. 20  shows sensor calibration at 10 mM. As shown in  FIGS. 17-19 , the sensor gives a linear and persistent response to D-3-hydroxybutyrate (ketone). Table  4  also shows the free NAD Ketone Sensor Beaker Calibration and Stability Summary. 
     
       
         
           
               
               
               
             
               
                   
                 TABLE 4 
               
               
                   
                   
               
             
            
               
                   
                 Slope 
                 2.55 
               
               
                   
                 R{circumflex over ( )}2 
                 0.9996 
               
               
                   
                 Decay in 45 hours 
                 −8% 
               
               
                   
                   
               
            
           
         
       
     
     EXAMPLE 3 
     Experiments were also performed to demonstrate the performance of ketone sensors having a working electrode that contains free NAD versus immobilized NAD. The sensors were prepared by depositing onto the surface of an electrode an enzyme composition containing the free NAD (A) or the immobilixed NAD (B). Sensing layer formulation is described in Table 5. The sensing layer solutions was deposit on carbon electrodes, and cured at 25C/60H overnight, prior to addition of membrane. The membrane formulation is provided in Table 6. The sensor was dipped (Table 7) from above solution 3×5 mm/sec, and the sensor was cured at 25C/60H overnight, 56C for two days.  FIG. 21  shows that both the free and immobilized NAD versions of the ketone sensor show similar stabilities and signals. 
     
       
         
           
               
             
               
                 TABLE 5 
               
             
            
               
                   
               
               
                 Sensing Layer Solution 
               
               
                 BD161116 
               
            
           
           
               
               
               
            
               
                 10 mM Hepes 
                 Mixing uL 
                 Final mg/mL 
               
            
           
           
               
               
               
               
               
               
               
               
            
               
                 D-3-Hydroxybutyrate 
                 Vendor 
                 Cat 
                 mg/mL 
                 A 
                 B 
                 A 
                 B 
               
               
                   
               
            
           
           
               
               
               
               
               
               
               
               
            
               
                 Dehydrogenase (HBD) 
                 Toyobo 
                 HBD-301 
                 40 
                 20 
                 20 
                 8.89 
                 8.89 
               
               
                 Diaphorase 
                 Toyobo 
                 DAD-311 
                 40 
                 20 
                 20 
                 8.89 
                 8.89 
               
               
                 NAD 
                 Sigma 
                 N0632 
                 20 
                 10 
                   
                 2.22 
                 0.00 
               
               
                 NAD-NH2 
                 Biotium 
                   
                 40 
                   
                 10 
                 0.00 
                 4.44 
               
               
                 Glutaraldehyde 
                   
                   
                 10 
                 0 
                 5 
                 0.00 
                 0.56 
               
               
                 Polymeric redox mediator 
                   
                   
                 40 
                 20 
                 20 
                 8.89 
                 8.89 
               
               
                 Peg400 
                   
                   
                 40 
                 20 
                 20 
                 8.89 
                 8.89 
               
               
                 ToTal 
                   
                   
                   
                 90 
                 95 
               
               
                   
               
            
           
         
       
     
     
       
         
           
               
             
               
                 TABLE 6 
               
               
                   
               
               
                 Membrane Coating 
               
               
                 Membrane Solution 
               
               
                   
               
             
            
               
                   
               
            
           
           
               
               
               
               
               
            
               
                 Solution A 
                 Vendor 
                 MW 
                 Mixing 
               
               
                   
               
               
                 Smart Membrane 
                 ADC 
                 160K 
                 120 
                 mg 
               
               
                 Ethanol/Hepe (10 mM pH 8.0) 
                   
                   
                 1 
                 mL 
               
               
                   
               
            
           
           
               
               
               
               
               
            
               
                 Solution B 
                 Vendor 
                 Cat 
                 Mixing 
               
               
                   
               
               
                 Poly(ethylene glycol) diglycidyl 
                 Polysciences 
                 8210 
                 100 
                 mg 
               
               
                 ether (PEGDGE 400) 
               
               
                 Ethanol/Hepe (10 mM pH 8.0) 
                   
                   
                 1 
                 mL 
               
               
                   
               
            
           
           
               
               
               
            
               
                   
                 Final membrane solution 
                 Mixing (mL) 
               
               
                   
                   
               
               
                   
                 Solution A 
                 4 
               
               
                   
                 Solution B 
                 0.35 
               
               
                   
                   
               
            
           
         
       
     
     
       
         
           
               
             
               
                 TABLE 7 
               
             
            
               
                   
               
               
                 Membrane Dipping Condition (Free NAD sensor was coated 
               
               
                 thicker membrane than Immobilized NAD sensor) 
               
            
           
           
               
               
               
            
               
                   
                 SL Solution 
                 Dipping 
               
               
                   
                   
               
               
                   
                 A (Free NAD) 
                 4 × 5 
               
               
                   
                 B (Immobilized NAD) 
                 3 × 5 
               
               
                   
                   
               
            
           
         
       
     
     EXAMPLE 4 
     In vitro and in vivo experiments were performed using a three-electrode sensor (i.e., working electrode, reference electrode, and counter electrode) to demonstrate the performance of a continuous ketone monitor calibrated using in vitro sensitivity. The sensor included the chemistry described in Example 1 above. The sensors were manufactured using methods to control the area of the sensing layer on the working electrode and to control the thickness of the membrane layer. All sensors used in this experiment were manufactured in the same lot. 
     In vitro testing was performed to determine in vitro sensitivity of a baseline subset of sensors from a manufacturing lot (in this case, 16 sensors), for example, as shown and described in connection with  FIG. 19  and Table 4 above. The baseline subset may include quantities other than sixteen, without departing from the scope of the present subject matter. The in vitro test sensitivity was obtained by applying various ketone solutions to each analyte sensor and monitoring the electrical current produced as a result, which can be on the order of nanoamps, picoamps, or otherwise depending on the sensor design. In vitro testing comprised placing and submerging the baseline subset of 16 sensors in a solution of 100 mM phosphate buffer at a controlled temperature of 37° C., sequentially forming a plurality of known ketone concentrations in the injecting into the solution aliquots of 1M ketone to achieve various ketone concentrations (for example, without limitation, 1, 2, 3, 4, 6, and 8 mmol/L in the solution), measuring the current from each sensor at the ketone concentration (i.e., ketone concentration of 1, 2, 3, 4, 6, and 8 mmol/L in the solution) with a potentiostat, and determining by performing a regression (e.g., linear or non-linear) independently on each respective in vitro test data set. As embodied herein, a plurality of known ketone concentration can include any range of ketone concentration of 1-8 mmol/L. 
     As can be seen in  FIG. 22 , from time 0 to time 0.2 hours, no solution is applied to the sensors (or a solution having no ketone concentration is applied). At time 0.2 a first ketone solution having a first relatively low concentration (e.g., one millimole per liter (mmol/L)) is applied to the sensor and the resulting response is recorded. At time 0.4 a second ketone solution having a relatively higher concentration than the first solution is applied to the sensor and the resulting response is again recorded. The process can proceed iteratively at 0.6 and thereafter with ever increasing concentrations of ketone solution to obtain empirical data representing the sensitivity of the ketone sensor across a wide range of ketone concentrations. As can be seen, these embodiments of the ketone sensors react differently to the presence of the ketone solution and these differences become more pronounced as the concentration of the ketone solution increases. Note that the x-axis indicates time and not ketone concentration, so while the in vitro test data may appear to be slightly nonlinear, the resulting sensitivity derived from the in vitro test data can still be linear. 
     In some embodiments, such as for nonlinear sensitivities, the in vitro data set can be portioned to separate response zones, with each zone being modeled with a linear sensitivity to approximate the nonlinear curve, such that the resulting calibration information will differ depending on the degree of response (e.g., current) being measured. As shown in  FIG. 16  and discussed in Example  1  above, the sensitivity is linear or substantially linear. The in vitro sensitivity (or other sensing characteristic) of the baseline subset can be determined in any desired fashion. In some embodiments, a number of different in vitro data subsets from the manufacturing lot can be used to determine a plurality of sensitivities, and the baseline in vitro sensitivity can be a central tendency of the plurality of determined sensitivities, such as a mean or median of sensitivities. In some embodiments, the baseline in vitro sensitivity can be a central tendency (e.g., mean or median) of one aspect or characteristic of sensitivities, such as the central tendency of the slopes of sensitivities or the central tendency of the intercepts of sensitivities. Other aspects of the sensitivities can also be used as the in vitro sensitivity for the baseline subset. In some embodiments, instead of deriving individual sensitivities from each of the in vitro test data sets, a single regression can be performed for the entirety of the in vitro test data from the baseline subset and this single regression, or an aspect thereof, can be used as the baseline in vitro sensitivity. In all of these embodiments, the in vitro test data sets or the in vitro sensing characteristics determined therefrom can be filtered to remove one or more values (e.g., values below a minimum threshold, above a maximum threshold, within a threshold, atypical values, etc.) prior to determining the baseline in vitro sensitivity. 
     In the illustrated example, in vitro sensor sensitivity was quantified by the slope of a least square regression through the current versus ketone concentration, as done for Example 1 and illustrated in  FIG. 19 . The in vitro sensitivity was used to generate calibrated sensor response for all in vitro studies. The calibrated sensor response generated by 16 sensors at 37° C. with sequential addition of ketone aliquots is presented in  FIG. 22  (solid line is the mean and shaded area is one standard deviation of the data from 16 sensors). The average coefficient of variation of the sensor response across the ketone levels is 5.0%. As can be seen in  FIG. 23 , the calibrated sensors show a linear response against the ketone concentration with a R2=0.9994. Importantly, as can be seen in  FIG. 23 , the linear response represents a slope of 1.0003, indicating that the sensor current calibrated using the determined in vitro sensitivity closely approximates ketone concentration in the solution. 
     Additionally, response time of the sensors was calculated as the time required for the sensor response to change from 10% above baseline and to 90% of the plateau for each aliquot addition. The sensors responded to the change in the ketone concentration within 4 minutes (average response time is 228 seconds) of adding the ketone aliquots to the test solution. 
     The stability of the 16 sensors over an exemplary intended wear period (for example, without limitation, 14 days) was assessed under simulated conditions. In particular, 16 sensors were submerged in phosphate buffer with 8 mM of ketone at 37° C. for 14 days. The operational stability of the sensors is presented in  FIG. 24  (solid line is the mean and shaded area is one standard deviation of the data from 16 sensors). Operational stability is critical for a ketone sensor, specifically because the sensor may not be calibrated by the user since the baseline ketone level is typically very low, unlike glucose. Achieving operational stability for over 14 days for a NAD+ dependent chemistry is even more challenging as NAD+ is a free molecule, difficult to be retained in the sensing chemistry. Additionally, stability of sensor response was measured by measuring the drift in the sensor response over the test period. As can be seen in  FIG. 24 , sensor signal at 8 mM was stable over the 14 days with an average daily signal loss of 0.15% (total signal loss over 14 days is 2.1%). As such, a sensor can be used with a single calibration for at least 14 days of use. Additionally, a drift correction factor can be determined for the entire lot of sensors in the manufacturing lot based on the drift measured during in vitro testing of the subset of in vivo sensors being tested in vitro. 
     Finally, interference from ascorbic acid was assessed by testing 10 sensors under in vitro conditions in phosphate buffer at 37° C. The sensors were tested at 0.6 mM and 1.5 mM of ketone in solution. After the sensor signal was stabilized, ascorbic acid was introduced to achieve an ascorbic acid concentration of 2 mg/dL, representing a level higher than the highest concentration under therapeutic treatment. The change in sensor response after the addition of ascorbic acid was measured The interference suggests that the sensor signal may change by no more than 0.2 mmol/L equivalent. This interference is independent of the concentration of ketone. 
     In addition, a clinical study was performed to evaluate in vivo performance of the sensors. 12 healthy volunteers were enrolled and required to be on a low carbohydrate diet and willing to remain on that diet throughout the study. The volunteers included 11 female and 1 male participants with an average age of 32.3 (range: 20 to 51) years. One of the participants had T1D. One of the participants was of Hispanic race while all other participants identified themselves as white. Average BMI was 24.3 (range: 18.6 to 30) kg/m2 with 7 of the 12 participants having a BMI of &lt;25 kg/m2. All participants self-reported as practicing a low carbohydrate diet. 
     Two sensors each were placed on the back of both upper arms of each study participant (i.e., a total of 4 sensors per participant). Three of these sensors were functional ketone sensors and one of the sensors used did not contain any functional chemistry (i.e., a total of 36 ketone sensors and 12 sensors without functional chemistry were used). Out of 36 ketone sensors and 12 background sensors tested in the study, 31 ketone sensors and 11 background sensors had evaluable data. Data from the failed 5 ketone sensors and 1 background sensor was excluded from data analysis. 
     The participants wore the sensors for up to 14 days. The sensors were activated using a reader device, such as those described herein, and sensors started measuring the signal 60 minutes after activation. All sensor results were masked to the study participants. The study participants were required to perform eight daily fingerstick measurements using Precision Xtra ketone test strips, over the waking period, preferably upon waking, before each meal, an hour after the meal and at bedtime. 
     The data of the non-functional sensors from all study participants were used to establish a single background current signal model independent of participant. According to embodiments, the background current signal may also be obtained by in-vitro methods such as those described herein, including, without limitaion, by applying various ketone solutions to each analyte sensor, without departing from the scope of the present subject matter. Signals from the functional sensors were first corrected with this background current signal before calculating the ketone results from the functional sensor. A retrospective calibration for each sensor was derived by correlating the sensor current to the reference values. A sensitivity value was determined for each capillary ketone measurement as a ratio of the sensor current (corrected for temperature) to the capillary ketone value, i.e., sensitivity=current/capillary ketone concentration. To simulate no calibration by the user, no further adjustments were made to evaluate the accuracy over 14 days. The sensitivity assigned to each sensor was the median of the individual sensitivity measurements for that sensor. Response of the three functional sensors over the 14 days to ketone levels in the body for one of the study participants is presented in  FIG. 25 . As can be seen in  FIG. 25 , all three sensors accurately track the capillary ketone reference through the entire 14 days of wear. 
     In sum, a total of 3128 paired datapoints were collected from the clinical study, which included in vivo sensor measurements and reference ketone measurements. The reference measurement ranged from 0-5.1 mM, with a median value of 0.6 mM. The current measured by the in vivo ketone sensors was calibrated using the baseline in vitro sensitivity determined previously based on a baseline subset of 16 in vivo sensors to determine sensor ketone measurements.  FIG. 26A  shows the correlation between sensor ketone measurements calibrated based on retrospective sensor calibration using methods described herein and ketone reference values. As can be seen in  FIG. 26A , calibrated sensor ketone measurements accurately reflect interstitial ketone levels, as illustrated by a slope of 0.908. Although  FIG. 26A  only illustrates a predictive relationship up to ketone values of 5.1 mM, a predictive relationship exists up to ketone values of approximately 6 mM. In some embodiments, a predictive relationship exists up to ketone values of approximately 8 mM, as can be seen in  FIGS. 26B-G . According to embodiments, in vivo ketone sensors can be calibrated using the determined drift correction factor, as described above, in addition to the determined in vitro sensitivity. 
     Furthermore, to assess accuracy of the calibrated sensor ketone results, sensor ketone results were compared to the capillary ketone reference results obtained by the Precision Xtra ketone test strips. At concentrations below 1.5 mM, the accuracy against the reference was calculated as mM and at or above 1.5 mM, it was calculated as percentage. The accuracy results are summarized in Table 8 below. For reference ketone concentrations &lt;1.5 mM, the overall MAD is 0.129 mM with 83.4% of points within +/−0.225 mM and 91.7% within +/−0.3 mM. For reference ketone concentrations &gt;=1.5 mM, the overall MARD is 14.4% with 76.0% within 20% and 89.7% within 30%. For the full range of concentrations, the values are 82.4% within 0.225 mM/20% and 91.4% within 0.3 mM/30%. 
     
       
         
           
               
               
               
               
             
               
                 TABLE 8 
               
               
                   
               
               
                   
                 Percentage 
                 Percentage 
                 Number of 
               
               
                 Concentration 
                 Within 
                 Within 
                 Paired data 
               
               
                 range 
                 0.225 mM/20% 
                 0.3 mM/30% 
                 points 
               
               
                   
               
             
            
               
                   
               
            
           
           
               
               
               
               
            
               
                  &lt;1.5 mM 
                 83.4% 
                 91.7% 
                 2720 
               
               
                 &gt;=1.5 mM 
                 76.0% 
                 89.7% 
                 408 
               
               
                 Combined 
                 82.4% 
                 91.4% 
                 3128 
               
               
                   
               
            
           
         
       
     
     According to embodiments disclosed herein, a system can comprise an in vivo ketone sensor having a distal portion configured for placement in contact with an interstitial fluid of a user and a proximal portion, a sensor control unit comprising at least one contact in electrical communication with the proximal portion of the sensor, and a transmitter configured to communicate with a remote device; wherein the sensor control unit is configured to receive the generated signals, and convert the generated signals to ketone concentration data using a sensitivity associated with the in vivo ketone sensor; and the transmitter is configured to communicate the ketone concentration data to the remote device. The sensor can comprising a working electrode, a sensing layer comprising β-hydroxybutyrate dehydrogenase, and a membrane layer configured to limit transport of one or more biomolecules, wherein the in vivo ketone sensor is configured to generate signals at the working electrode corresponding to an amount of ketone in the interstitial fluid. 
     All features, elements, components, functions, and steps described with respect to any embodiment provided herein are intended to be freely combinable and substitutable with those from any other embodiment. If a certain feature, element, component, function, or step is described with respect to only one embodiment, then it should be understood that that feature, element, component, function, or step can be used with every other embodiment described herein unless explicitly stated otherwise. This paragraph therefore serves as antecedent basis and written support for the introduction of claims, at any time, that combine features, elements, components, functions, and steps from different embodiments, or that substitute features, elements, components, functions, and steps from one embodiment with those of another, even if the following description does not explicitly state, in a particular instance, that such combinations or substitutions are possible. It is explicitly acknowledged that express recitation of every possible combination and substitution is overly burdensome, especially given that the permissibility of each and every such combination and substitution will be readily recognized by those of ordinary skill in the art. 
     In all of the embodiments described herein, electronic devices capable of processing data or information can include processing circuitry communicatively coupled with non-transitory memory, where the non-transitory memory can store one or more computer program or software instructions that, when executed by the processing circuitry, cause the processing circuitry to take actions. For every embodiment of a method disclosed herein, systems and devices capable of performing those methods, or portions thereof, with processing circuitry and non-transitory memory having one or more instructions stored thereon that, when executed by the processing circuitry, cause that processing circuitry to execute one or more steps of the method (or cause the execution of one or more steps of the method, such as transmission or display of information), are within the scope of the present disclosure. 
     Computer program or software instructions for carrying out operations in accordance with the described subject matter may be written in any combination of one or more programming languages, including an object oriented programming language such as Java, JavaScript, Smalltalk, C++, C#, Transact-SQL, XML, PHP or the like and conventional procedural programming languages, such as the “C” programming language or similar programming languages. The program instructions may execute entirely on the computing device, partly on the computing device, as a stand-alone software package, partly on a local computing device and partly on a remote computing device or entirely on a remote computing device or server. In the latter scenario, the remote computing device may be connected to the local computing device through any type of network, including a local area network (LAN) or a wide area network (WAN), or the connection may be made to an external computer (for example, through the Internet using an Internet Service Provider). 
     To the extent the embodiments disclosed herein include or operate in association with memory, storage, and/or computer readable media, then that memory, storage, and/or computer readable media are non-transitory. Accordingly, to the extent that memory, storage, and/or computer readable media are covered by one or more claims, then that memory, storage, and/or computer readable media is only non-transitory. 
     As used herein and in the appended claims, the singular forms “a,” “an,” and “the” include plural referents unless the context clearly dictates otherwise. 
     While the embodiments are susceptible to various modifications and alternative forms, specific examples thereof have been shown in the drawings and are herein described in detail. It should be understood, however, that these embodiments are not to be limited to the particular form disclosed, but to the contrary, these embodiments are to cover all modifications, equivalents, and alternatives falling within the spirit of the disclosure. Furthermore, any features, functions, steps, or elements of the embodiments may be recited in or added to the claims, as well as negative limitations that define the inventive scope of the claims by features, functions, steps, or elements that are not within that scope.