WO2015178912A1

WO2015178912A1 - Analyte sensors and methods of using same

Info

Publication number: WO2015178912A1
Application number: PCT/US2014/039175
Authority: WO
Inventors: Tianmei Ouyang; Benjamin J. Feldman; Lam N. Tran; Yi Wang
Original assignee: Abbott Diabetes Care Inc.
Priority date: 2014-05-22
Filing date: 2014-05-22
Publication date: 2015-11-26

Abstract

Provided are sensors for determining the concentration of an analyte in a sample fluid. In certain embodiments, the sensors include semiconducting particles and exhibit improved uniformity of distribution of one or more sensing chemistry components, increased effective working electrode surface area and improved fill time consistency. Methods of using and manufacturing the sensors are also provided.

Description

ANALYTE SENSORS AND METHODS OF USING SAME

INTRODUCTION

[0001] In many instances it is desirable or necessary to regularly monitor the

concentration of particular constituents in a fluid. A number of systems are available that analyze the constituents of bodily fluids such as blood, urine and saliva. Examples of such systems conveniently monitor the level of particular medically significant fluid constituents, such as, for example, cholesterol, ketones, vitamins, proteins, and various metabolites or blood sugars, such as glucose. Diagnosis and management of patients suffering from diabetes, a disorder where either the pancreas produces insufficient insulin which prevents normal regulation of blood sugar levels or cells do not respond to the insulin that is produced, requires carefully monitoring of blood glucose levels on a daily basis. A number of systems that allow individuals to easily monitor their blood glucose are currently available. Such systems include electrochemical biosensors, including those that comprise a glucose sensor that is adapted to determine the concentration of an analyte in a bodily fluid (e.g., blood) sample.

[0002] A person may obtain a blood sample by withdrawing blood from a blood source in his or her body, such as a vein, using a needle and syringe, for example, or by lancing a portion of his or her skin, using a lancing device, for example, to make blood available external to the skin, to obtain the necessary sample volume for in vitro testing. The person may then apply the fresh blood sample to a test strip, whereupon suitable detection methods, such as colorimetric, electrochemical, or photometric detection methods, for example, may be used to determine the person's actual blood glucose level.

[0003] Analyte sensors with improved performance, such as increased accuracy and response times, are desirable. The present disclosure provides sensors, and methods of using and manufacturing such sensors, meeting these and a variety of other needs.

SUMMARY

[0004] Provided are sensors for determining the concentration of an analyte in a sample fluid. In certain embodiments, the sensors include semiconducting particles and exhibit improved uniformity of distribution of one or more sensing chemistry components, increased effective working electrode surface area and improved reliability without short circuiting the electrodes. In certain embodiments, the improved reliability is obtained while reducing the reagent chemistry used per sensor, thereby reducing costs. In another embodiment, semiconducting particles on a surface of the sample chamber improve sample fill time consistency. Methods of using and manufacturing the sensors are also provided.

[0005] These and other objects, advantages, and features of the invention will become apparent to those persons skilled in the art upon reading the details of the embodiments as more fully described below.

BRIEF DESCRIPTION OF THE FIGURES

[0006] The invention is best understood from the following detailed description when read in conjunction with the accompanying drawings. It is emphasized that, according to common practice, the various features of the drawings are not to-scale. On the contrary, the dimensions of the various features are arbitrarily expanded or reduced for clarity. Included in the drawings are the following figures.

[0007] FIG. 1 is a microphotograph of deposition of a conventional sensing layer formulation on an electrode surface, wherein the sensing layer does not include semiconducting particles.

[0008] FIG. 2 is a microphotograph of deposition of a sensing layer formulation on an electrode surface, wherein the sensing layer includes semiconducting particles.

[0009] FIGS. 3A-3C show a schematic view of non-uniform reagent distribution upon drying of a detection reagent solution in the sample chamber of a sensor.

[0010] FIG. 4 is a microphotograph of deposition of a conventional sensing layer formulation on an electrode surface, wherein the sensing layer does not include semiconducting particles with the electrode surface sectioned.

[0011] FIG. 5 is a schematic view of a first embodiment of a sensor strip in accordance with the present disclosure.

[0012] FIG. 6 is an exploded perspective view of the sensor strip shown in FIG. 5 with the layers illustrated individually with the electrodes in a first configuration.

[0013] FIG. 7 is an exploded perspective view of a second embodiment of a sensor strip in accordance with the present disclosure with the layers illustrated individually with the electrodes in a second configuration. [0014] FIG. 8 is a comparison graph of the active electrode area of a control strip, a strip having a conventional sensing layer formulation, and a strip having a sensing layer formulation including semiconducting particles of the present disclosure.

[0015] FIG. 9 is a comparison graph of fill times for strips having a conventional sensing layer formulation, and strips having a sensing layer formulation including semiconducting particles of the present disclosure at different temperatures.

[0016] FIG. 10 is a comparison graph of the coefficient of variation for strips having a conventional sensing layer formulation, and strips having a sensing layer formulation including semiconducting particles of the present disclosure at different temperatures.

[0017] FIG. 11A is a graph of bias from control across various glucose levels for a conventional sensing layer versus a sensing layer formulation including semiconducting particles of the present disclosure.

[0018] FIG. 1 IB is a graph of fill time across various glucose levels for the conventional sensing layer of FIG. 11 A versus a sensing layer formulation including semiconducting particles of the present disclosure.

[0019] FIG. 12A is a graph of charge for strips having different loadings of a

conventional sensing layer formulation across various glucose levels.

[0020] FIG. 12B is a graph of charge for strips having different loadings of a sensing layer formulation including semiconducting particles of the present disclosure across various glucose levels.

[0021] FIG. 13A is a graph illustrating the precision of a conventional sensing layer.

[0022] FIG. 13B is a graph illustrating the precision of a sensing layer formulation including semiconducting particles of the present disclosure.

DETAILED DESCRIPTION

[0023] Before the sensors and methods of the present disclosure are described in greater detail, it is to be understood that the sensors and methods are not limited to particular

embodiments described, as such may, of course, vary. It is also to be understood that the terminology used herein is for the purpose of describing particular embodiments only, and is not intended to be limiting, since the scope of the sensors and methods will be limited only by the appended claims. [0024] Where a range of values is provided, it is understood that each intervening value, to the tenth of the unit of the lower limit unless the context clearly dictates otherwise, between the upper and lower limit of that range and any other stated or intervening value in that stated range, is encompassed within the sensors and methods. The upper and lower limits of these smaller ranges may independently be included in the smaller ranges and are also encompassed within the sensors and methods, subject to any specifically excluded limit in the stated range. Where the stated range includes one or both of the limits, ranges excluding either or both of those included limits are also included in the sensors and methods.

[0025] Certain ranges are presented herein with numerical values being preceded by the term "about." The term "about" is used herein to provide literal support for the exact number that it precedes, as well as a number that is near to or approximately the number that the term precedes. In determining whether a number is near to or approximately a specifically recited number, the near or approximating unrecited number may be a number which, in the context in which it is presented, provides the substantial equivalent of the specifically recited number.

[0026] Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which the methods belong. Although any methods and materials similar or equivalent to those described herein can also be used in the practice or testing of the sensors and methods, representative illustrative sensors, methods and materials are now described.

[0027] All publications and patents cited in this specification are herein incorporated by reference as if each individual publication or patent were specifically and individually indicated to be incorporated by reference and are incorporated herein by reference to disclose and describe the sensors, methods and/or materials in connection with which the publications are cited. The citation of any publication is for its disclosure prior to the filing date and should not be construed as an admission that the present sensors and methods are not entitled to antedate such publication by virtue of prior invention. Further, the dates of publication provided may be different from the actual publication dates which may need to be independently confirmed.

[0028] It is noted that, as used herein and in the appended claims, the singular forms "a",

"an", and "the" include plural referents unless the context clearly dictates otherwise. It is further noted that the claims may be drafted to exclude any optional element. As such, this statement is intended to serve as antecedent basis for use of such exclusive terminology as "solely," "only" and the like in connection with the recitation of claim elements, or use of a "negative" limitation.

[0029] It is appreciated that certain features of the sensors and methods, which are, for clarity, described in the context of separate embodiments, may also be provided in combination in a single embodiment. Conversely, various features of the sensors and methods, which are, for brevity, described in the context of a single embodiment, may also be provided separately or in any suitable sub-combination. All combinations of the embodiments are specifically embraced by the present invention and are disclosed herein just as if each and every combination was individually and explicitly disclosed, to the extent that such combinations embrace operable processes and/or devices/systems/kits. In addition, all sub-combinations listed in the

embodiments describing such variables are also specifically embraced by the present sensors and methods and are disclosed herein just as if each and every such sub-combination was

individually and explicitly disclosed herein.

[0030] As will be apparent to those of skill in the art upon reading this disclosure, each of the individual embodiments described and illustrated herein has discrete components and features which may be readily separated from or combined with the features of any of the other several embodiments without departing from the scope or spirit of the present sensors and methods. Any recited method can be carried out in the order of events recited or in any other order which is logically possible.

OVERVIEW

[0031] Provided are sensors for determining the concentration of an analyte in a sample fluid. In certain embodiments, the sensors include semiconducting particles and exhibit improved uniformity of distribution of one or more sensing chemistry components and increased effective working electrode surface area without short circuiting the electrodes. The improved uniformity of distribution and increased effective working electrode surface area result in improved linearity, improved reproducibility, improved yield, improved reaction time, improved glucose recovery and reduced cost. Methods of using and manufacturing the sensors are also provided. The components are disposed proximate to a working electrode of the sensor, such as in vivo and/or in vitro analyte sensors, including, continuous and/or automatic in vivo analyte sensors. For example, embodiments herein provide for inclusion of semiconducting particles in a solution, such as a sensing layer formulation. Also provided are systems and methods of using the analyte sensors in analyte monitoring.

[0032] In general, embodiments disclosed herein are based on the discovery that the addition of semiconducting particles to solution formulations used in the manufacture of in vivo and/or in vitro biosensors greatly improves uniformity and/or distribution of one or more reagent components of the sensor (e.g., an enzyme-containing sensing layer of such devices) as compared to a sensor lacking the semiconducting particles. The result is a reduction, and in some cases the complete elimination, of the buildup of the reagent solution along the edges of the strip, known as the "coffee ring" effect (see homogenous and uniform distribution of the sample in FIG. 2 as compared to the example in FIG. 1). This results in a more uniform distribution of the constituents of a solution deposited on a substrate upon drying and curing, as well as a smoother surface of the solution upon drying and curing as compared to a solution lacking the

semiconducting particles. The uniform distribution improves reagent efficiency, thereby reducing the amount of reagent used.

[0033] Embodiments disclosed herein are also based on the discovery that disposition of semiconducting particles on a surface of the working electrode of the sensor, such as in vitro or in vivo analyte sensors, results in a working electrode with increased effective surface area while reducing or eliminating short circuits in the electrode.

[0034] Moreover, the addition of semiconducting particles to solution formulations of such sensors also results in an improved linearity of the sensor over a range of reagent loading as compared to a conventional sensor lacking the semiconducting particles. In addition, in certain embodiments, the semiconducting particles provide for improved fill times as well as improved accuracy over time and over a range of temperatures.

[0035] The semiconducting particles may be included in any component of a sensor that can benefit from improvement of uniformity of distribution and/or increased electrode surface area. Exemplary components include, but are not limited to, formulations that provide reagents in a sensing layer having an analyte responsive enzyme.

[0036] Additional exemplary components of a sensor that may be suitably formulated with semiconducting particles are described in U.S. Pat. Nos. 5,262,035, 5,262,305, 6,134,461, 6,143,164, 6,175,752, 6,338,790, 6,579,690, 6,654,625, 6,736,957, 6,746,582, 6,932,894, 6,605,200, 6,605,201, 7,090,756, 6,746,582, 8,255,034 as well as those described in U.S. Patent Publication No. 2008/0179187, all of which are incorporated herein by reference in their entirety.

[0037] Embodiments of the present disclosure relate to sensors having improved uniformity of distribution of one or more analyte detection reagents by inclusion of

semiconducting particles in the sensor solution, where the detection reagent and semiconducting particles are disposed on a surface of a working electrode of the sensor, such as in vitro or in vivo analyte sensors. For example, embodiments of the present disclosure provide for inclusion of semiconducting particles in a solution, such as a detection reagent solution, resulting in more uniform distribution of the detecting reagent after the detection reagent solution is dried on the surface of the working electrode. Also provided are methods of manufacturing the analyte sensors and methods of using the analyte sensors in analyte monitoring.

SENSORS

Sensor with Uniform Reagent Distribution

[0038] In certain aspects, during the manufacturing process for the subject analyte sensors, a detection reagent solution is contacted with a surface of a substrate (e.g., a surface of a working electrode), forming a deposition of the solution on the surface of the substrate. In some cases, the solution is allowed to dry and cure. One difficulty with producing analyte sensors in which the reagent solution is deposited in a channel (e.g., a channel constituting a sample chamber region defined by a spacer layer of the sensor) is that - as the reagent solution dries - the reagent tends to deposit preferentially at the sides of the channel. In such instances, the reagent will not be uniformly distributed across the channel (e.g., along the axis perpendicular to the flow of a sample into the sample chamber), and the center of the channel may be partially denuded of reagent, resulting in the analyte not reacting completely in the center of the channel, particularly at high analyte concentrations (e.g., high blood glucose concentrations). This non- uniformity may adversely affect performance characteristics of the sensor, such as decreasing accuracy and increasing fill time as compared to a sensor having substantially uniform distribution of the detection reagent on the working electrode surface.

[0039] The result of non-uniform reagent distribution as a reagent solution dries in a sample chamber of a sensor is illustrated in FIG. 1 and is schematically shown in FIGS. 3A-3C. A portion of a partially assembled sensor includes sample chamber 102 defined by substrate 104 and spacer layer 106. As shown in FIGS. 3B and 3C, as reagent solution dries, the reagent (shown as dots) preferentially deposits at sides 108 of the sample chamber 102. FIG. 3C shows a top view of the portion of the sensor shown in FIG. 3B.

[0040] FIG. 4 illustrates the actual mal-distribution of the reagent on the surface of a sensor. For example, a rectangular working electrode (with, e.g., a deposited enzyme) of width 1.5 mm and length 4 mm, is scribed into 3 smaller rectangular sub-areas A, B and C, each 0.5 mm by 4 mm. Each sub-area is then washed with buffer to extract the deposited enzyme into a known volume of liquid, e.g., about 1 mL. Enzyme activity assays are used to quantify the amount of enzyme in each sub-area. The working electrode in the sample chamber of FIG. 4 is scribed into sub-area A, sub-area B and sub-area C. Sub-area A measured 1.45 units of enzyme, sub-area B measured 0.34 units of enzyme and sub-area C measured 1.31 units of enzyme.

[0041] Embodiments of the present disclosure are based on the discovery that the addition of semiconducting particles to a reagent solution used in the manufacture of in vitro or in vivo analyte sensors improves uniformity and/or distribution of one or more detection reagents (e.g., an analyte-responsive enzyme and/or redox mediator) on a surface of the sensor. The results of adding semiconducting particles to a reagent solution for improved reagent uniformity and distribution is schematically illustrated in FIG. 2. The reagent solution is disposed in the sample chamber and includes detection reagent(s) and semiconducting particles. As the reagent solution dries, the semiconducting particles inhibit the preferential deposition of reagent components at the sides of the sample chamber. As shown in FIG. 2, the result is a dry reagent semiconducting particles composition disposed on the substrate (e.g., a working electrode surface) in the sample chamber region, the composition having substantially uniform distribution of the detection reagent(s) attributable to the semiconducting particles being present in the reagent solution during the drying.

[0042] It will be appreciated that the particle density of the reagent solution/suspension may be adjusted to achieve a desired amount/configuration of particles in the sample chamber upon drying of the solution/suspension. For example, the particle density may be chosen to achieve uniform distribution of the analyte detection reagent, and also to provide less than a single layer of particles on the surface of the substrate. In other aspects, the particle density may be chosen to achieve uniform distribution of the analyte detection reagent, and also to provide a monolayer, bilayer, or more layers of the particles on the surface of the substrate. [0043] Accordingly, in certain embodiments, the present disclosure provides sensors for determining the concentration of an analyte (e.g., glucose, a ketone, etc.) in a sample fluid. The sensors include a first substrate having a proximal end and a distal end, the first substrate defining a first side edge and a second side edge of the sensor extending from the proximal end to the distal end of the first substrate, the distal end being configured and arranged for insertion into a sensor reader. According to this aspect, the sensors also include a second substrate disposed over the first substrate, a working electrode disposed on one of the first and second substrates, a counter electrode disposed on one of the first and second substrates, and a spacer disposed between the first and second substrates and defining a sample chamber that comprises the working electrode and the counter electrode. Also according to this aspect, the sensors include a dry composition disposed on an area of the working electrode, the composition comprising an analyte detection reagent and semiconducting particles configured and arranged to provide for substantially uniform distribution of the detection reagent on the area of the working electrode.

[0044] By "substantially uniform distribution" of the detection reagent is meant that if the area of the working electrode upon which the dry composition is disposed was subdivided into equal sized smaller areas (or "sub-areas"), these sub-areas would each have the same or substantially the same quantity of disposed analyte detection reagent, within the uncertainty of the measurement method used to quantify the disposed reagent. In certain embodiments, the quantity of analyte detection reagent disposed on the sub-areas does not differ between sub-areas by more than about 25%. For example, the quantity of detection reagent disposed on the sub- areas does not differ between sub-areas by more than about 20%, 15%, 10%, 5%, 2%, or more than about 1%.

[0045] The area of the working electrode on which the composition is disposed may comprise any desired amount of semiconducting particles. In certain aspects, the area of the working electrode on which the dry composition is disposed includes between about 0.001 and

10 mg of particles per cm . For example, the area of the working electrode may include between about 0.01 and 5 mg of particles per cm semiconducting particles/mm2, between about 0.02 and

2 mg of particles per cm semiconducting particles particles/mm2, between about 0.04 and 1 mg of particles per cm , or between about 0.08 and 0.16 mg of particles per cm semiconducting particles/mm2. The particle density may depend, e.g., on the diameter of the particles, the concentration of the particles in the "wet" composition applied to the working electrode surface prior to drying, and/or the like.

Sensors having Increased Electroactive Surface Area

[0046] Embodiments of the present disclosure relate to sensors having a working electrode with increased effective surface area by disposition of semiconducting particles on a surface of the working electrode of the sensor, such as in vitro or in vivo analyte sensors. Also provided are methods of manufacturing the analyte sensors and methods of using the analyte sensors in analyte monitoring.

[0047] The inventors of the present disclosure have found that disposing one or more layers of semiconducting particles on a surface of a working electrode increases the effective surface area of the working electrode. As a result of the increased effective surface area of the working electrode, higher peak currents, shorter test times, and/or more linear results may be obtained during operation of the sensor. Strips with semiconducting particles included with the reagent solution show a significant increase in double layer charging compared with both a blank strip and a strip having the reagent solution only.

[0048] It had been thought that using particles that were not conductive particles would reduce the working electrode surface area, as non-conducting particles may limit access to the working electrode surface area and particles having low conductivity may simply result in a working electrode having a surface area with decreased effectiveness. However, the inventors of the present disclosure have found that the semiconducting particles have a conductivity that is sufficiently high to extend the electrode surface rather than reduce or eliminate the electrode surface area, while also having a conductivity that is too low to cause short circuits, even when the semiconducting particles bridge the short space between facing working and counter electrodes. Sensor strips containing semiconducting particles were deliberately miss-coated outside of the channel of the sample chamber and tested. Even with semiconducting particles outside of the channel, short circuiting was not observed.

[0049] The working electrode surface can have a monolayer of semiconducting particles disposed thereon. The semiconducting particles are in contact with the working electrode surface, as well as with each other, such that each particle acts as an extension of the electrode surface. In other aspects, provided are sensors having a working electrode surface with more than one layer of semiconducting particles disposed thereon. In this aspect, semiconducting particles of a first layer makes contact with the working electrode surface, with each other, and also with semiconducting particles of a second layer. The semiconducting particles of the second layer make contact with the semiconducting particles of the first layer, as well as each other. Whether one, two or more layers of semiconducting particles are provided on the surface of the working electrode, the result is that all or a majority of the particles act as an extension of the working electrode surface, thereby increasing the effective surface area of the working electrode.

[0050] According to certain embodiments, one or more layers of semiconducting particles are disposed on a surface of the working electrode (e.g., by depositing a semiconducting particle-containing suspension on the working electrode surface, followed by drying), and an analyte detection solution that includes one or more analyte detection reagents (e.g., an analyte- responsive enzyme and/or a redox mediator) is deposited over the semiconducting particles. In other aspects, one or more layers of semiconducting particles are disposed on the surface of the working electrode by applying a semiconducting particle suspension that also includes one or more analyte detection reagents (e.g., an analyte-responsive enzyme and/or a redox mediator).

[0051] The desired number of layers of semiconducting particles on the working electrode surface may be achieved using any suitable approach. For example, when a particle- containing suspension is deposited on a working electrode surface, the particle density of the suspension may be controlled to achieve a monolayer, bilayer, tri-layer, or more layers of semiconducting particles on the surface. Whether or not a particular particle density achieves the desired number of layers may be determined, e.g., via microscopic imaging of the particles disposed on the electrode surface. In certain aspects, the desired number of layers of

semiconducting particles is achieved by first determining the particle density of a particle suspension required to achieve a monolayer of particles on the electrode surface (for particles having a particular diameter), and then increasing the particle density by a factor of two, three or more to achieve a bilayer, tri-layer, or more layers, respectively, of semiconducting particles on the surface.

[0052] Other approaches for achieving the desired number of particle layers on the working electrode surface include inkjet deposition and spray deposition, both of which can be performed in multiple passes, such that the preceding pass is allowed to dry before a subsequent pass/layer is applied. The semiconducting particles may be deposited over the entire surface of the working electrode. Alternatively, the surface of the working electrode on which the semiconducting particles are deposited is substantially limited to all or a portion of the working electrode surface positioned within the sample chamber of the sensor.

[0053] Additional advantages are realized with certain embodiments of the present disclosure of sensors having improved uniformity of distribution of one or more analyte detection reagents and/or increased effective working electrode surface area due to the incorporation of semiconducting particles.

Sensors having Signal Enhancement

[0054] In certain embodiments of the present disclosure, the inclusion of semiconducting particles on a working electrode surface result in sensors with an increased signal, i.e., peak current. A higher signal is indicative of both well distributed reagents and increased

electroactive area. Sensors having higher signals from the inclusion of semiconducting particles on a working electrode surface may result in shortened sensor response times.

[0055] In certain cases, the analyte sensor comprising semiconducting particles on a working electrode surface provides a signal from electrolysis of an analyte in a sample that is at least 5%, or 10%, or 15%, or 20%, or 30%, or 40%, or 50%, or 60%, or 70%, or 80%, or 90%, or 100%, or 120%, or 130% higher than the signal generated from electrolysis of the analyte in the sample using a similar analyte sensor but not comprising semiconducting particles on a working electrode surface. In certain embodiments, the higher signal is generated within 0.01 second, or 0.03 second, or 0.01 second, or 0.3 second, or 0.6 second, or 1 second, or 1.3 seconds, or 1.6 seconds, or 2 seconds, or 3 seconds, or 4 seconds, or 5 seconds, or more of applying the sample to the analyte sensor. As used herein, "signal" refers to current, charge, resistance, voltage, impedance, or log or integrated values thereof that is related to the concentration of the analyte being analyzed by the sensor.

Sensors having Stable Fill Time

[0056] In certain embodiments of the present disclosure of sensors having improved uniformity of distribution of one or more analyte detection reagents and/or increased effective working electrode surface area due to the incorporation of semiconducting particles, fill time of the sample chamber shows greater stability over time and over different temperature ranges. [0057] In certain cases, the analyte sensor comprising semiconducting particles provides a fill time after eight weeks that is at least 5%, or 10%, or 15%, or 20%, or 30%, or 40%, or 50%, or 60%, or 70%, or 80%, or 90%, or 100%, or 110%, or 120%, or 130%, or 140%, or 150%, or 160%, or 170%, or 180%, or 190%, or 200% or more lower than the fill time generated in the sample using a similar analyte sensor but not comprising semiconducting particles.

Therefore, the use of semiconducting particles improves the fill time and thus the shelf life of the strips over a wide range of temperatures.

[0058] According to certain embodiments, one or more layers of semiconducting particles are disposed on a surface of the sample chamber (e.g., by depositing a semiconducting particle-containing suspension on the desired surface of the sample chamber, followed by drying). The semiconducting particles may be disposed on a portion or all of the sample chamber surface area to improve the fill time and thus the shelf life of the strips. For example, the semiconducting particles can be disposed on all surfaces of the sample chamber. As another example, the semiconducting particles can be disposed on surfaces of the sample chamber excluding one or both of the working electrode and the counter electrode. In other aspects, one or more layers of semiconducting particles are disposed on the surface of the working electrode by applying a semiconducting particle suspension that also includes one or more analyte detection reagents (e.g., an analyte-responsive enzyme and/or a redox mediator).

[0059] The desired number of layers of semiconducting particles on the surface of the sample chamber may be achieved using any suitable approach. For example, when a particle- containing suspension is deposited on a surface, the particle density of the suspension may be controlled to achieve a monolayer, bilayer, tri-layer, or more layers of semiconducting particles on the surface. Whether or not a particular particle density achieves the desired number of layers may be determined, e.g., via microscopic imaging of the particles disposed on the surface. In certain aspects, the desired number of layers of semiconducting particles is achieved by first determining the particle density of a particle suspension required to achieve a monolayer of particles on the electrode surface (for particles having a particular diameter), and then increasing the particle density by a factor of two, three or more to achieve a bilayer, tri-layer, or more layers, respectively, of semiconducting particles on the surface.

[0060] Other approaches for achieving the desired number of particle layers on the surface include inkjet deposition and spray deposition, both of which can be performed in multiple passes, such that the preceding pass is allowed to dry before a subsequent pass/layer is applied. The semiconducting particles may be deposited over the entire surface of the working electrode.

Sensors having Increased Reproducibility

[0061] In certain embodiments of the present disclosure of sensors having improved uniformity of distribution of one or more analyte detection reagents and/or increased effective working electrode surface area due to the incorporation of semiconducting particles, the sensors exhibit increased reproducibility (accuracy) over time and over different temperature ranges.

[0062] In certain cases, the analyte sensor comprising semiconducting particles exhibits reproducibility at a rate at least 5%, or 10%, or 15%, or 20%, or 30%, or 40%, or 50%, or 60%, or 70%, or 80%, or 90%, or 100%, or 110%, or 120%, or 130%, or 140%, or 150%, or 160%, or 170%, or 180%, or 190%, or 200% or more greater than the reproducibility exhibited by a sample using a similar analyte sensor but not comprising semiconducting particles. As reproducibility increases, yield increases. Accuracy of the strips overtime is also improved.

Sensors having Increased Linearity

[0063] In certain embodiments of the present disclosure of sensors having improved uniformity of distribution of one or more analyte detection reagents and/or increased effective working electrode surface area due to the incorporation of semiconducting particles, the sensors exhibit increased linearity over a range of loading.

[0064] In certain cases, the analyte sensor comprising semiconducting particles exhibits linearity at least across reagent loadings from 40% to 100% and across analyte levels from 50 to 350 mg/dL. The charge for the analyte sensor comprising semiconducting particles remained nearly identical at a particular glucose level at least 40%, at least 60%, at least 80% and 100%.

[0065] The analyst sensor without semiconducting particles exhibit non-linearity at all reagent loading levels. The non-linearity is a product of the uneven reagent deposition on the working electrode when no semiconducting particles are used. As reagent loading is decreased to 40%, this non-linearity increases substantially. The charge for the analyte sensor without semiconducting particles dropped significantly at a particular glucose level from 100% to 40%. [0066] The analyte sensors comprising the semiconducting particles exhibit superior recovery at the various loadings as compared with the strip without semiconducting particles at 100% loading, indicating that reagent loading can be reduced with the use of semiconducting particles without sacrificing charge.

[0067] Exemplary features and components of the sensors provided by the present disclosure are described in greater detail below. It will be appreciated that any such features and components may be employed - or present in - any of the sensors described above, including sensors having improved uniformity of distribution of one or more analyte detection reagents and/or increased effective working electrode surface area. A variety of analyte sensor

configurations are known in the art which may be suitable for use in the disclosed embodiments. Additional suitable sensor and meter configurations include, but are not limited to, those described in U.S. Patent Publication No. 2011/0287528 and U.S. Patent No. 6,616,819.

[0068] Referring to FIG. 5 and FIG. 6, a first embodiment of a sensor 10 is schematically illustrated, herein shown in the shape of a strip. It is to be understood that the sensor may be any suitable shape. Sensor strip 10 has a first substrate 12, a second substrate 14, and a spacer 15 positioned there between. Sensor strip 10 includes at least one working electrode 24 and at least one counter electrode 22. Sensor strip 10 also includes an optional insertion monitor 30. Sensor strip 10 has a first, proximal end 10A and an opposite, distal end 10B. At proximal end 10A, the sample to be analyzed is applied to sensor 10. Proximal end 10A could be referred as 'the fill end', 'sample receiving end', or similar. Distal end 10B of sensor 10 is configured for operable, and usually releasable, connecting to a device such as a meter. Sensor strip 10 is a layered construction, in certain embodiments having a generally rectangular shape, i.e., its length is longer than its width, although other shapes are possible as well, as noted above. The length of sensor strip 10 is from end 10A to end 10B.

[0069] The dimensions of a sensor may vary. In certain embodiments, the overall length of sensor strip 10 may be no less than about 10 mm and no greater than about 50 mm. For example, the length may be between about 30 and 45 mm; e.g., about 30 to 40 mm. It is understood, however that shorter and longer sensor strips 10 could be made. In certain embodiments, the overall width of sensor strip 10 may be no less than about 3 mm and no greater than about 15 mm. For example, the width may be between about 4 and 10 mm, about 5 to 8 mm, or about 5 to 6 mm. In one particular example, sensor strip 10 has a length of about 32 mm and a width of about 6 mm. In another particular example, sensor strip 10 has a length of about 40 mm and a width of about 5 mm. In yet another particular example, sensor strip 10 has a length of about 34 mm and a width of about 5 mm.

[0070] As provided above, sensor strip 10 has first and second substrates 12, 14, nonconducting, inert substrates which form the overall shape and size of sensor strip 10. Substrates 12, 14 may be substantially rigid or substantially flexible. In certain embodiments, substrates 12, 14 are flexible or deformable. Examples of suitable materials for substrates 12, 14 include, but are not limited, to polyester, polyethylene, polycarbonate, polypropylene, nylon, and other "plastics" or polymers. In certain embodiments the substrate material is "Melinex" polyester. Other non-conducting materials may also be used.

[0071] Substrate 12 includes first or proximal end 12A and second or distal end 12B, and substrate 14 includes first or proximal end 14A and second or distal end 14B. As indicated above, positioned between substrate 12 and substrate 14 may be spacer 15 to separate first substrate 12 from second substrate 14. In some embodiments, spacer 15 extends from end 10A to and 10B of sensor strip 10, or extends short of one or both ends. Spacer 15 is an inert nonconducting substrate, typically at least as flexible and deformable (or as rigid) as substrates 12, 14. In certain embodiments, spacer 15 is an adhesive layer or double-sided adhesive tape or film that is continuous and contiguous. Any adhesive selected for spacer 15 should be selected to not diffuse or release material which may interfere with accurate analyte measurement. In certain embodiments, the thickness of spacer 15 may be constant throughout, and may be at least about 0.01 mm (10 μιη) and no greater than about 1 mm or about 0.5 mm. For example, the thickness may be between about 0.02 mm (20 μιη) and about 0.2 mm (200 μιη). In one certain

embodiment, the thickness is about 0.05 mm (50 μιη), and about 0.1 mm (100 μιη) in another embodiment.

[0072] The sensor includes a sample chamber for receiving a volume of sample to be analyzed; in the embodiment illustrated, particularly in FIG. 5, sensor strip 10 includes sample chamber 20 having an inlet 21 for access to sample chamber 20. In the embodiment illustrated, sensor strip 10 is a side-fill sensor strip, having inlet 21 present on a side edge of strip 10. Tip-fill sensors, having an inlet at, for example, end 10A, are also within the scope of this disclosure and are described with reference to Fig. 7, as well as corner and top filling sensors. Sample chamber 20 is configured so that when a sample is provided in chamber 20, the sample is in electrolytic contact with both a working electrode and a counter electrode, which allows electrical current to flow between the electrodes to affect the electrolysis (electro-oxidation or electro-reduction) of the analyte. Sample chamber 20 is defined by substrate 12, substrate 14 and spacer 15; in many embodiments, sample chamber 20 exists between substrate 12 and substrate 14 where spacer 15 is not present. Typically, a portion of spacer 15 is removed to provide a volume between substrates 12, 14 without spacer 15; this volume of removed spacer is sample chamber 20. For embodiments that include spacer 15 between substrates 12, 14, the thickness of sample chamber 20 is generally the thickness of spacer 15.

[0073] Sample chamber 20 has a volume sufficient to receive a sample of biological fluid therein. In some embodiments, such as when sensor strip 10 is a small volume sensor, sample chamber 20 has a volume that is typically no more than about 1 μί, for example no more than about 0.5 μί, and also for example, no more than about 0.25 μL·. A volume of no more than about 0.1 μΐ_^ is also suitable for sample chamber 20, as are volumes of no more than about 0.05 μϊ_^ and about 0.03 μL·. The thickness of sample chamber 20 corresponds typically to the thickness of spacer 15. Particularly for facing electrode configurations, as in the sensor illustrated in FIG. 5, this thickness is small to promote rapid electrolysis of the analyte, as more of the sample will be in contact with the electrode surface for a given sample volume. In addition, a thin sample chamber 20 helps to reduce errors from diffusion of analyte into the sample chamber during the analyte assay, because diffusion time is long relative to the measurement time, which may be about 5 seconds or less.

[0074] As provided above, the sensor includes a working electrode and at least one counter electrode. The counter electrode may be a counter/reference electrode. If multiple counter electrodes are present, one of the counter electrodes will be a counter electrode and one or more may be reference electrodes. The sensor includes at least one working electrode positioned within the sample chamber. In FIG. 6, working electrode 24 is illustrated on substrate 14. In alternate embodiments, a working electrode is present on a different surface or substrate, such as substrate 12. Working electrode 24 extends from the sample chamber 20, proximate first end 10A, to the other end of the sensor 10, end 10B, as an electrode extension called a "trace". The trace provides a contact pad 25 for providing electrical connection to a meter or other device to allow for data and measurement collection, as will be described later. Contact pad 25 may be positioned on a tab 27 that extends from the substrate on which working electrode 24 is positioned, such as substrate 12 or 14. In some embodiments, a tab has more than one contact pad positioned thereon. In alternate embodiments, a single contact pad is used to provide a connection to one or more electrodes; that is, multiple electrodes are coupled together and are connected via one contact pad.

[0075] As provided above, at least a portion of working electrode 24 is provided in sample chamber 20 for the analysis of analyte, in conjunction with the counter electrode.

Referring to FIG. 6 and in accordance with one embodiment of the present disclosure, a dry composition including at least one analyte detection reagent (e.g., an analyte-responsive enzyme and/or a redox mediator) and semiconducting particles may be disposed on a surface of an area of working electrode 24, e.g., all or a portion of the surface of working electrode region 32. The dry composition may be disposed on working electrode 24 prior to disposing spacer 15 on substrate 14. Alternatively, spacer 15 is first disposed on substrate 14 (creating a channel having working electrode 24 as its base, similar to the channel shown in FIG. 3A), followed by application of an aqueous composition that includes at least one detection reagent and semiconducting particles to all or a portion of the surface of working electrode region 32. In accordance with the present disclosure, as the aqueous composition dries, the detection reagent(s) remain distributed substantially uniformly over the working electrode surface due to the presence of the semiconducting particles in the composition (see, e.g., FIG. 2).

[0076] In another embodiment of the present disclosure, at least one analyte detection reagent may be disposed on a surface of the working electrode 24, e.g., all or a portion of the surface of working electrode region 32. Semiconducting particles may be disposed on a surface of the sample chamber 20, e.g., all or a portion of one or more than one surface of the sample chamber 20. The sample chamber 20 is defined by substrate 12, substrate 14 and spacer 15; so the semiconducting particles may be disposed on one or more of the surfaces of substrate 12, substrate 14, spacer 15 that form the sample chamber 20. As the working electrode 24 is positioned on substrate 12 or substrate 14 and the counter electrode 22 is positioned on substrate 12 or substrate 14, the semiconducting particles can be disposed on at least a portion of one or both of the working electrode 24 and the counter electrode 22.

[0077] For sensor 10, at least one counter electrode is positioned on one of first substrate

12 and second substrate 14 in the sample chamber. In FIG. 6, counter electrode 22 is illustrated on substrate 12. Counter electrode 22 extends from the sample chamber 20, proximate end 10A, to the other end of the sensor 10, end 10B, as an electrode extension called a "trace". The trace provides a contact pad 23 for providing electrical connection to a meter or other device to allow for data and measurement collection, as will be described later. Contact pad 23 may be positioned on a tab 26 that extends from the substrate on which counter electrode 22 is positioned, such as substrate 12. In some embodiments, a tab has more than one contact pad positioned thereon. In alternate embodiments, a single contact pad is used to provide a connection to one or more electrodes; that is, multiple electrodes are coupled together and are connected via one contact pad.

[0078] Working electrode 24 and counter electrode 22 may be disposed opposite to and facing each other to form facing electrodes. See for example, FIG. 6, which has working electrode 24 on substrate 14 and counter electrode 22 on substrate 12, forming facing electrodes. In this configuration, the sample chamber is typically present between the two electrodes 22, 24. Working electrode 24 and counter electrode 22 may alternately be positioned generally planar to one another, such as on the same substrate, to form co-planar or planar electrodes.

[0079] In some instances, it is desirable to be able to determine when the sample chamber of the sensor is sufficiently filled with sample. Sensor strip 10 may be indicated as filled, or substantially filled, by observing a signal between an optional indicator electrode and one or both of working electrode 24 or counter electrode 22 as sample chamber 20 fills with fluid. When fluid reaches the indicator electrode, the signal from that electrode will change. Suitable signals for observing include, for example, voltage, current, resistance, impedance, or capacitance between the indicator electrode and, for example, working electrode 24.

Alternatively, the sensor may be observed after filling to determine if a value of the signal (e.g., voltage, current, resistance, impedance, or capacitance) has been reached indicating that the sample chamber is filled. The optional indicator electrode may also be used to improve the precision of the analyte measurements. The indicator electrode may operate as a working electrode or as a counter electrode or counter/reference electrode. Measurements from the indicator electrode/working electrode may be combined (e.g., added or averaged) with those from the first counter/reference electrode/working electrode to obtain more accurate

measurements. [0080] The sensor or equipment that the sensor is connected with (e.g., a meter) may include a signal (e.g., a visual sign or auditory tone) that is activated in response to activation of the indicator electrode to alert the user that the sample chamber is beginning to fill with sample and/or that the sample chamber is sufficiently filled with sample to measure the analyte concentration. The sensor or equipment may be configured to initiate a reading when the indicator electrode indicates that the sample chamber has been filled with or without alerting the user. The reading may be initiated, for example, by applying a potential between the working electrode and the counter electrode and beginning to monitor the signals generated at the working electrode.

[0081] Referring to FIG. 7, another embodiment of an analyte sensor is illustrated as analyte sensor 210. The analyte sensor strip 210 has a first substrate 212, a second substrate 214, and a spacer 215 positioned there between. Analyte sensor strip 210 includes at least one working electrode 222 and at least one counter electrode 224. Analyte sensor strip 210 has a first, proximal end and an opposite, distal end. At proximal end, sample to be analyzed is applied to sensor 210. Proximal end could be referred as "the fill end" or "sample receiving end". Distal end of sensor 210 is configured for operable connection to a device such as a meter. Sensor strip 210 is a layered construction, in certain embodiments having a generally rectangular shape, which is formed by first and second substrates 212, 214. Substrate 212 includes first or proximal end 212A and second or distal end 212B, and substrate 214 includes first or proximal end 214A and second or distal end 214B.

[0082] Sensor strip 210 includes sample chamber 220 having an inlet 221 for access to sample chamber 220. Sensor strip 210 is a tip-fill sensor, having inlet 221 at the proximal end. Sample chamber 220 is defined by substrate 212, substrate 214 and spacer 215. Generally opposite to inlet 221, through substrate 212 is a vent 230 from sample chamber 220.

[0083] For sensor 210, at least one working electrode 222 is illustrated on substrate 214.

Working electrode 222 extends from end 214A into sample chamber 220 to end 214B. Sensor 210 also includes at least one counter electrode 224, in this embodiment on substrate 214.

Counter electrode 224 extends from sample chamber 220, proximate first proximal end to distal end. Working electrode 222 and counter electrode 224 are present on the same substrate, e.g., as planar or co-planar electrodes. [0084] Referring to FIG. 7 and in accordance with an embodiment of the present disclosure, a dry composition including at least one analyte detection reagent (e.g., an analyte- responsive enzyme and/or a redox mediator) and semiconducting particles may be disposed on a surface of an area of working electrode 222, e.g., all or a portion of the surface of working electrode area 236. The dry composition may be disposed on working electrode 222 prior to disposing spacer 215 on substrate 214. Alternatively, spacer 215 is first disposed on substrate 214 (creating a channel having working electrode 222 as a portion of its base, similar to the channel shown in FIG. 3A), followed by application of an aqueous composition that includes at least one detection reagent and semiconducting particles to an exposed portion of the surface of working electrode area 232. In accordance with the present disclosure, as the aqueous composition dries, the at least one detection reagent remains distributed substantially uniformly over the working electrode surface to which the composition is applied, due to the presence of the semiconducting particles in the composition (see, e.g., the sensor in FIG. 2).

[0085] In another embodiment of the present disclosure, at least one analyte detection reagent may be disposed on a surface of the working electrode 224, e.g., all or a portion of the surface of working electrode region 232. Semiconducting particles may be disposed on a surface of the sample chamber 220, e.g., all or a portion of one or more than one surface of the sample chamber 220. The sample chamber 220 is defined by substrate 212, substrate 214 and spacer 215; so the semiconducting particles may be disposed on one or more of the surfaces of substrate 212, substrate 214, spacer 215 that form the sample chamber 220. As the working electrode 224 is positioned on substrate 212 or substrate 214 and the counter electrode 222 is positioned on substrate 212 or substrate 214, the semiconducting particles can be disposed on at least a portion of one or both of the working electrode 224 and the counter electrode 222.

Working Electrode

[0086] As summarized previously herein, an analyte sensor includes a working electrode and a reference/counter electrode, comprising a first portion located in the sample chamber and a second portion for connection to a meter. The working electrode may be formed from a suitable conducting material. The conducting material may have relatively low electrical resistance and may be electrochemically inert over the potential range of the sensor during operation and substantially transparent. In certain aspects, the working electrode includes a material selected from the group consisting of gold, carbon, platinum, ruthenium, palladium, silver, silver chloride, silver bromide, and combinations thereof. The working electrode may be a thin layer of gold, tin oxide, platinum, ruthenium dioxide or palladium, indium tin oxide, zinc oxide, fluorine doped tin oxide, as well as other non-corroding materials known to those skilled in the art. The working electrode can be a combination of two or more conductive materials. For example, the working electrode may be constructed from thin layer of gold in the sample chamber and of carbon outside the sample chamber.

[0087] The working electrode can be applied on a substrate by any of a variety of methods, including by being deposited, such as by vapor deposition or vacuum deposition or otherwise sputtered, printed on a flat surface or in an embossed or otherwise recessed surface, transferred from a separate carrier or liner, etched, or molded. Suitable methods of printing include screen-printing, piezoelectric printing, ink jet printing, laser printing, photolithography, painting, gravure roll printing, transfer printing, and other known printing methods.

Semiconducting Particles

[0088] Semiconducting particles for improving the distribution of analyte detection reagents and increasing the effective surface area of a working electrode while avoiding short circuiting may be used in the sensors of the present disclosure.

[0089] In certain embodiments, the semiconducting particles can comprise, as non- limiting examples, tin oxide and antimony doped tin oxide. The semiconducting particles can be in solution, such as antimony doped tin oxide sol. The semiconducting particles can be dispersed, for example, in water or any other liquid suitable for water-based applications. Other non-limiting examples of semiconducting particles include Ge, CuCl, CuBr, Cul, AgCl, AgBr, Agl, Ag₂S, ZnS, HgS, ZnSe, CdS, CdSe, CdTe, HgTe, PbS, BN, A1N, GaN, Al_xGa xN, GaP GaAs, GaSb, InP, InAs, In_xGa xAs, Si xGe_x, Si₃N₄, ZrN, CaF₂, YF₃, A1₂0₃, Si0₂, Ti0₂, Zr₂0₃, Zr0₂, Sn0₂, YSi₂, GaInP₂, Cd₃P₂, Fe₂S, CuIn₂S₂, MoS₂, In₂S₃, Bi₂S₃, CuIn₂Se₂, In₂Se₃, Hgl₂ and Pbl₂.

[0090] The semiconducting particles can be a nanomaterial, such as a nanopowder or a nanoparticle. In such embodiments, the nanopowder or nanoparticle will have semiconducting particles having a diameter of from about 1 nm to about 300 nm, including about 10 nm to about 290 nm, about 15 nm to about 275 nm, about 20 nm to about 250 nm, about 30 nm to about 225 nm, about 35 nm to about 200 nm, about 40 nm to about 175 nm, about 45 nm to about 150 nm, about 50 nm to about 125 nm, about 55 nm to about 100 nm, and about 60 nm to about 75 nm. In certain aspects, the semiconducting particles are nanospheres or microspheres.

[0091] The semiconducting particles suitable for use in the embodiments disclosed will have conductivity within a range that provides the advantages discussed, including extending the electrode surface, while having conductivity too low to short circuit the electrodes. As a non- limiting example, antimony doped tin oxide semiconducting particles have a conductivity of approximately 12,000 ohm/sqr/mil.

Counter Electrode

[0092] The counter electrode may be constructed in a manner similar to the working electrode. As used herein, the term "counter electrode" refers to an electrode that functions as a counter electrode, or both a reference electrode and a counter electrode. The counter electrode can be formed, for example, by depositing electrode material onto a substrate. The material of the counter electrode may be deposited by a variety of methods such as those described above for the working electrode. In certain aspects, the counter electrode includes a material selected from the group consisting of gold, carbon, platinum, ruthenium, palladium, silver, silver chloride, silver bromide, and combinations thereof. For example, suitable materials for the counter electrode include Ag/AgCl or Ag/AgBr printed on a non-conducting substrate. According to certain embodiments, the counter electrode comprises a thin conductive layer such as gold, tin oxide, indium tin oxide, layered with AgCl or AgBr, for example.

Electrode Configuration

[0093] A variety of analyte sensor electrode configurations are known in the art which may be suitable for use in the disclosed analyte sensors. For example, suitable configurations can include configurations having a working electrode positioned in opposition to a

reference/counter electrode or configurations having the working electrode positioned coplanar with the reference/counter electrode. Additional suitable electrode configurations include, but are not limited to, those described in U.S. Patent Application Publication No. 2007/0095661; U.S. Patent Application Publication No. 2006/0091006; U.S. Patent Application Publication No. 2006/0025662; U.S. Patent Application Publication No. 2008/0267823; U.S. Patent Application Publication No. 2007/0108048; U.S. Patent Application Publication No. 2008/0102441; U.S. Patent Application Publication No. 2008/0066305; U.S. Patent Application Publication No. 2007/0199818; U.S. Patent Application Publication No. 2008/0148873; U.S. Patent Application Publication No. 2007/0068807; U.S. Patent Application Publication No. 2009/0095625; U.S. Patent No. 6,616,819; U.S. Patent No. 6,143,164; U.S. Patent No. 6,592,745; U.S. Patent No. 7,866,026; and U.S. Patent No. 8,262,874; the disclosures of each of which are incorporated herein by reference in their entireties for all purposes.

Analytes

[0094] A variety of analytes can be detected and quantified using the analyte sensors disclosed herein including, but not limited to, glucose, blood β-ketone, ketone bodies, lactate, acetyl choline, amylase, bilirubin, cholesterol, chorionic gonadotropin, creatine kinase (e.g., CK- MB), creatine, DNA, fructosamine, glutamine, growth hormones, hormones, ketones, lactate, peroxide, prostate-specific antigen, prothrombin, RNA, thyroid stimulating hormone, and troponin, in sample of body fluid.

[0095] Analyte sensors may also be configured to detect and/or quantify drugs, such as, for example, antibiotics (e.g., gentamicin, vancomycin, and the like), digitoxin, digoxin, drugs of abuse, theophylline, and warfarin. Assays suitable for determining the concentration of DNA and/or RNA are disclosed in U.S. Patent No. 6,281,006 and U.S. Patent No. 6,638,716, the disclosures of each of which are incorporated by reference herein.

Analyte-Responsive Enzyme

[0096] The disclosed analyte sensors may include in the sample chamber an analyte responsive enzyme which is capable of transferring electrons to or from a redox mediator and the analyte. For example, a glucose oxidase (GOD) or glucose dehydrogenase (GDH) can be used when the analyte is glucose. A lactate oxidase can be used when the analyte is lactate. These enzymes catalyze the electrolysis of an analyte by transferring electrons between the analyte and the electrode via the redox mediator.

[0097] In one embodiment, the analyte-responsive enzyme is disposed on the working electrode. In certain embodiments, the analyte-responsive enzyme is immobilized on the working electrode. This is accomplished, for example, by cross linking the analyte-responsive enzyme with a redox mediator on the working electrode, thereby providing a sensing layer on the working electrode. In an alternative embodiment, the analyte-responsive enzyme is disposed adjacent to the electrode.

[0098] Generally, the analyte-responsive enzyme and redox mediator are positioned in close proximity to the working electrode in order to provide for electrochemical communication between the analyte-responsive enzyme and redox mediator and the working electrode.

Generally, the analyte-responsive enzyme and redox mediator are positioned relative to the reference/counter electrode such that electrochemical communication between the analyte- responsive enzyme and the redox mediator and the reference/counter electrode is minimized.

[0099] Additional analyte-responsive enzymes and cofactors which may be used in connection with the disclosed analyte sensors are described in U.S. Patent No. 6,736,957 and U.S. Patent Publication No. 2011/0318810, the disclosures of which are incorporated by reference herein. As a non-liming example, a flavin-binding glucose dehydrogenase with a high substrate specificity for D-glucose can be used. The flavin-binding glucose dehydrogenase is derived from a microorganism belonging to the genus Mucor. The flavin-binding glucose dehydrogenase has a low reactivity for maltose, D-galactose and D-xylose compared to its reactivity for D-glucose, and therefore is relatively unaffected by these saccharide compounds. The flavin-binding glucose dehydrogenase is also relatively unaffected by dissolved oxygen, and allows accurate measurement of glucose amounts even in the presence of saccharide compounds other than glucose in samples.

[00100] Examples of preferred enzymes as flavin-binding glucose dehydrogenase enzymes are those having the following enzymo-chemical properties:

(1) exhibiting glucose dehydrogenase activity in the presence of an electron acceptor;

(2) having a molecular weight of the polypeptide chain portion of the protein of approximately 80 kDa;

(3) having low reactivity for maltose, D-galactose and D-xylose, with respect to reactivity for D-glucose;

(4) having a pH of 6.5-7.0;

(5) having an optimum temperature of 37-40° C;

(6) having residual activity of at least 80% after heat treatment at 40° C for 15 minutes; (7) using a flavin compound as coenzyme; and

(8) having a Km value of 26-33 mM for D-glucose.

[00101] Glucose dehydrogenase having such enzymo-chemical properties allows accurate measurement of D-glucose levels without being affected by saccharide compounds such as maltose, D-galactose and D-xylose present in measuring samples. Furthermore, because it has satisfactory activity in a pH range and temperature range that are suitable for clinical diagnosis such as measurement of blood glucose levels, it can be suitably used as a diagnostic

measurement reagent or the like.

[00102] The property parameters mentioned above are typical examples, but these parameters have permissible variable ranges within limits allowing the effect of the invention to be achieved when measurement of D-glucose is conducted under prescribed measuring conditions.

[00103] In some embodiments, in order to facilitate the electrochemical reaction of the analyte sensor the sample chamber also includes an enzyme co-factor. For example, where the analyte-responsive enzyme is glucose dehydrogenase (GDH), suitable cofactors include pyrroloquinoline quinone (PQQ), nicotinamide adenine dinucleotide NAD+ and flavin adenine dinucleotide (FAD). In certain embodiments, the analyte detected and/or measured by the sensor described herein may be ketone and the enzyme included in the sensor is hydroxybutyrate dehydrogenase.

Redox Mediator

[00104] In addition to the analyte-responsive enzyme, the sample chamber may include a redox mediator. In one embodiment, the redox mediator is immobilized on the working electrode. Materials and methods for immobilizing a redox mediator on an electrode are provided in U.S. Patent No. 6,592,745, the disclosure of which is incorporated by reference herein. In an alternative embodiment, the redox mediator is disposed adjacent to the working electrode.

[00105] The redox mediator mediates a current between the working electrode and the analyte when present. The mediator functions as an electron transfer agent between the electrode and the analyte. Almost any organic or organometallic redox species can be used as a redox mediator. In general, suitable redox mediators are rapidly reducible and oxidizable molecules having redox potentials a few hundred millivolts above or below that of the standard calomel electrode (SCE), and typically not more reducing than about -200 mV and not more oxidizing than about +400mV versus SCE.

[00106] Examples of organic redox species are quinones and quinhydrones and species that in their oxidized state have quinoid structures, such as Nile blue and indophenol.

Unfortunately, some quinones and partially oxidized quinhydrones react with functional groups of proteins such as the thiol groups of cysteine, the amine groups of lysine and arginine, and the phenolic groups of tyrosine which may render those redox species unsuitable for some of the sensors of the present invention, e.g., sensors that will be used to measure analyte in biological fluids such as blood.

[00107] In certain cases, mediators suitable for use in the analyte sensors have structures which prevent or substantially reduce the diffusional loss of redox species during the period of time that the sample is being analyzed. Suitable redox mediators include a redox species bound to a polymer which can in turn be immobilized on the working electrode. Useful redox mediators and methods for producing them are described in U.S. Patent Nos. 5,262,035; 5,264,104;

5,320,725; 5,356,786; 6,592,745; and 7,501,053, the disclosure of each of which is incorporated by reference herein.

[00108] Any organic or organometallic redox species can be bound to a polymer and used as a redox mediator. In certain cases, the redox species is a transition metal compound or complex. The transition metal compounds or complexes may be osmium, ruthenium, iron, and cobalt compounds or complexes. In certain cases, the redox mediator may be an osmium compounds and complex. One type of non-releasable polymeric redox mediator contains a redox species covalently bound in a polymeric composition. An example of this type of mediator is poly(vinylferrocene) .

[00109] Alternatively, a suitable non-releasable redox mediator contains an

ionicallybound redox species. Typically, these mediators include a charged polymer coupled to an oppositely charged redox species. Examples of this type of mediator include a negatively charged polymer such as Nafion® (Dupont) 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 such as quaternized poly(4-vinyl pyridine) or poly(l -vinyl imidazole) coupled to a negatively charged redox species such as ferricyanide or ferrocyanide. [00110] In another embodiment, the suitable non-releasable redox mediators include a redox species coordinatively bound to the polymer. For example, the mediator may be formed by coordination of an osmium or cobalt 2, 2'-bipyridyl complex to poly(l -vinyl imidazole) or poly(4-vinyl pyridine).

[00111] The redox mediator may be a osmium transition metal complex with one or more ligands having a nitrogen-containing heterocycle such as 2,2' -bipyridine, 1,10-phenanthroline or derivatives thereof. Furthermore, the redox mediator may also have one or more polymeric ligands having at least one nitrogen-containing heterocycle, such as pyridine, imidazole, or derivatives thereof. These mediators exchange electrons rapidly between each other and the electrodes so that the complex may be rapidly oxidized and reduced.

[00112] In particular, it has been determined that osmium cations complexed with two ligands containing 2,2' -bipyridine, 1,10-phenanthroline, or derivatives thereof, the two ligands not necessarily being the same, and further complexed with a polymer having pyridine or imidazole functional groups form particularly useful redox mediators in the small volume sensors. Derivatives of 2,2' -bipyridine for complexation with the osmium cation may be 4,4'- dimethyl-2,2' -bipyridine and mono-, di-, and polyalkoxy-2,2'-bipyridines, such as 4,4'- dimethoxy-2,2' -bipyridine, where the carbon to oxygen ratio of the alkoxy groups is sufficient to retain solubility of the transition metal complex in water.

[00113] Preferred derivatives of 1,10-phenanthroline for complexation with the osmium cation are 4,7-dimethyl- 1,10-phenanthroline and mono-,di-, and polyalkoxy-1,10- phenanthrolines, such as 4,7-dimethoxy- 1,10-phenanthroline, where the carbon to oxygen ratio of the alkoxy groups is sufficient to retain solubility of the transition metal complex in water. Exemplary polymers for complexation with the osmium cation include poly(l-vinyl imidazole), e.g., PVI, and poly(4- vinyl pyridine), e.g., PVP, either alone or with a copolymer. Most preferred are redox mediators with osmium complexed with poly(l -vinyl imidazole) alone or with a copolymer.

[00114] Suitable redox mediators have a redox potential between about -150 mV to about

+400 mV versus the standard calomel electrode (SCE). For example, the potential of the redox mediator can be between about -100 mV and +100 mV, e.g., between about -50 mV and +50 mV. In one embodiment, suitable redox mediators have osmium redox centers and a redox potential more negative than +100 mV versus SCE, e.g., the redox potential is more negative than +50 mV versus SCE, e.g., is near -50 mV versus SCE.

[00115] In one embodiment, the redox mediators of the disclosed analyte sensors are airoxidizable. This means that the redox mediator is oxidized by air, e.g., so that at least

90% of the mediator is in an oxidized state prior to introduction of sample into the sensor. Airoxidizable redox mediators include osmium cations complexed with two mono-, di-, or polyalkoxy-2,2'-bipyridine or mono-, di-, or polyalkoxy-l,10-phenanthroline ligands, the two ligands not necessarily being the same, and further complexed with polymers having pyridine and imidazole functional groups. In particular, Os[4,4'-dimethoxy-2,2'-bipyridine]2Cl+/+2 complexed with poly(4- vinyl pyridine) or poly(l-vinyl imidazole) attains approximately 90% or more oxidation in air. In one specific embodiment, the redox mediator is 1,10 Phenanthrolene- 5,6-dione (PQ).

[00116] To prevent electrochemical reactions from occurring on portions of the working electrode not coated by the mediator, a dielectric may be deposited on the electrode surrounding the region with the bound redox mediator. Suitable dielectric materials include waxes and nonconducting organic polymers such as polyethylene. Dielectric may also cover a portion of the redox mediator on the electrode. The covered portion of the mediator will not contact the sample, and, therefore, will not be a part of the electrode's working surface.

[00117] Although it can be advantageous to minimize the amount of redox mediator used, the range for the acceptable amount of redox mediator typically has a lower limit. The minimum amount of redox mediator that may be used is the concentration of redox mediator that is necessary to accomplish the assay within a desirable measurement time period, for example, no more than about 5 minutes, or no more than about 1 minute, or no more than about 30 seconds, or no more than about 10 seconds, or no more than about 5 seconds, or no more than about 3 seconds, or no more than about 1 second or less.

[00118] The analyte sensor can be configured (e.g., by selection of redox mediator, positioning of electrodes, etc.) such that the sensor signal is generated at the working electrode with a measurement period of no greater than about 5 minutes and such that a background signal that is generated by the redox mediator is no more than five times a signal generated by oxidation or reduction of 5mM analyte. In some embodiments, the analyte sensor is configured such that the background signal that is generated by the redox mediator is less than the signal generated by oxidation or reduction of 5mM glucose. In some embodiments, the background that is generated by the redox mediator is no more than 25% of the signal generated by oxidation or reduction of 5mM analyte, e.g., no more than 20%, no more than 15% or no more than 5%. In certain embodiments, the analyte is glucose and the background that is generated by the redox mediator is no more than 25% of the signal generated by oxidation or reduction of 5mM glucose, e.g., no more than 20%, no more than 15% or no more than 5% of the signal generated by electrolysis of glucose.

Sorbent Material

[00119] The sample chamber may be empty prior to entry of the sample. Optionally, the sample chamber can include a sorbent material to sorb and hold a fluid sample during detection and/or analysis. Suitable sorbent materials include polyester, nylon, cellulose, and cellulose derivatives such as nitrocellulose. The sorbent material facilitates the uptake of small volume samples by a wicking action which may complement or replace any capillary action of the sample chamber. In addition or alternatively, a portion or the entirety of the wall of the sample chamber may be covered by a surfactant, such as, for example, Zonyl FSO.

[00120] In some embodiments, the sorbent material is deposited using a liquid or slurry in which the sorbent material is dissolved or dispersed. The solvent or dispersant in the liquid or slurry may then be driven off by heating or evaporation processes. Suitable sorbent materials include, for example, cellulose or nylon powders dissolved or dispersed in a suitable solvent or dispersant, such as water. The particular solvent or dispersant should also be compatible with the material of the electrodes (e.g., the solvent or dispersant should not dissolve the electrodes).

[00121] One of the functions of the sorbent material is to reduce the volume of fluid needed to fill the sample chamber of the analyte sensor. The actual volume of sample within the sample chamber is partially determined by the amount of void space within the sorbent material. Typically, suitable sorbents consist of about 5% to about 50% void space. In one embodiment, the sorbent material consists of about 10% to about 25% void space. Fill Assist

[00122] The analyte sensors can be configured for top-filling, tip-filling, corner-filling, and/or side-filling. In some embodiments, the analyte sensors include one or more optional fill assist structures, e.g., one or more notches, cut-outs, indentations, and/or protrusions, which facilitate the collection of the fluid sample. For example, the analyte sensor can be configured such that the proximal end of the analyte sensor is narrower than the distal end of the analyte sensor. In one such embodiment, the analyte sensor includes a tapered tip at the proximal end of the analyte sensor, e.g., the end of the analyte sensor that is opposite from the end that engages with a meter.

[00123] Additional fill assist structures are described in U.S. Patent Publication No.

2008/0267823, the disclosure of which is incorporated by reference herein; and U.S. Patent No. 7,866,026, the disclosure of which is incorporated by reference herein.

METHODS OF DETERMINING ANALYTE CONCENTRATION

[00124] The sensors described herein find use in methods for determining the

concentration of an analyte in a fluid sample from a subject. Generally, these methods include contacting a fluid sample with the sensor, generating a sensor signal at the working electrode, and determining the concentration of the analyte using the sensor signal. It will be understood that the subject methods may employ any of the sensors described herein, e.g., sensors having improved uniformity of distribution of one or more analyte detection reagents and/or increased effective working electrode surface area.

[00125] A variety of approaches may be employed to determine the concentration of the analyte. In certain aspects, an electrochemical analyte concentration determining approach is used. For example, determining the concentration of the analyte using the sensor signal may be performed by coulometric, amperometric, potentiometric, or any other convenient

electrochemical detection technique.

[00126] According to certain embodiments, the subject methods include obtaining the sample from a subject. When the sample is a blood sample, the sample may be obtained, e.g., using a lancet to create an opening in a skin surface at which blood subsequently presents. The blood sample may be obtained from the finger of a subject. Alternatively, the blood sample may be obtained from a region of the subject having a lower nerve end density as compared to a finger. Obtaining a blood sample from a region having a lower nerve end density as compared to a finger is generally a less painful approach for obtaining a blood sample and may improve patient compliance, e.g., in the case of a diabetes patient where regular monitoring of blood glucose levels is critical for disease management.

METHODS OF MAKING ANALYTE SENSORS

[00127] Also provided by the present disclosure are methods of manufacturing analyte sensors. In certain aspects, methods are provided that include forming a working electrode on a first substrate, forming a spacer layer on the first substrate, the spacer layer defining a sample chamber region on the first substrate, and applying a reagent composition on a surface of the working electrode in the sample chamber region. The reagent composition may include one or more analyte detection reagents (e.g., an analyte-responsive enzyme and/or a redox mediator) and semiconducting particles, where the semiconducting particles provide for even distribution of the detection reagent(s) as the reagent composition dries on the working electrode. The sample chamber region on the first substrate includes at least a portion of a working electrode surface. Generally, the reagent composition is applied to all or a portion of the working electrode surface in the sample chamber region, thereby generating a modified working electrode surface in the sample chamber region on which one or more analyte detection reagents are substantially uniformly distributed.

[00128] Optionally, the methods further comprise disposing a counter electrode on the first substrate (e.g., on region of the first substrate distinct from the region on which the working electrode is disposed), or alternatively, disposing a counter electrode on a second substrate to be overlayed on the first substrate (thereby generating a facing electrode pair). Manufacturing the sensor is generally completed by overlaying a second substrate on the spacer layer and singulating individual sensors (e.g., by dye cutting, etc.) from the starting substrate material. General approaches for manufacturing analyte sensors are known in the art and are described, e.g., in U.S. Patent No. 7,866,026 and U.S. Patent No. 6,592,745, the full disclosures of which are incorporated herein by reference in their entirety for all purposes.

[00129] The present disclosure also provides methods of manufacturing analyte sensors, which methods include forming a working electrode on a first substrate, and disposing one or more layers of conductive material (e.g., conductive microspheres) on the working electrode, where the one or more layers make up an ordered array of semiconducting particles. By using semiconducting particles having a consistent size and shape, the semiconducting particles are capable of being stacked into ordered arrays, with easily tailored surface area and void volume. For example, the number of layers of semiconducting particles may be selected to provide a desired effective surface area of the working electrode (e.g., where the effective surface area can be increased or decreased by increasing or decreasing the number of layers of semiconducting particles, respectively). Alternatively, or additionally, the size distribution of the semiconducting particles may be selected to provide a desired void volume within the array of conductive microspheres.

[00130] Also provided are methods of manufacturing analyte sensors, which methods include forming a working electrode on a first substrate, and disposing one or more layers of semiconducting particles on the working electrode, where a number of layers of the one or more layers of semiconducting particles is selected to provide a desired effective surface area of the working electrode. Optionally, the one or more layers make up an ordered array of

semiconducting particles. The size distribution of the semiconducting particles may be selected to provide a desired void volume within the one or more layers of semiconducting particles.

[00131] In addition, the present disclosure provides of manufacturing analyte sensors, which methods include forming a working electrode on a first substrate, and disposing one or more layers of semiconducting particles on the working electrode, where a size distribution of the semiconducting particles is selected to provide a desired void volume within the one or more layers of semiconducting particles. The number of layers of the one or more layers of semiconducting particles may be selected to provide a desired effective surface area of the working electrode. The one or more layers optionally make up an ordered array of

semiconducting particles.

UTILITY

[00132] The subject sensors and methods find use in a variety of different applications where, e.g., the accurate determination of an analyte concentration by an analyte sensor is desired. For example, the methods are useful for obtaining and accurately determining the concentration of one or more analytes in a bodily sample, e.g., a blood sample. EXPERIMENTAL

[00133] The following examples are offered by way of illustration and not by way of limitation.

EXAMPLE 1: SENSORS WITH SEMICONDUCTING PARTICLES OF

ANTIMONY DOPED TIN OXIDE (ATO)

[00134] In the present example, ATO semiconducting particles were obtained as a ca. 20%

(w/v) solution, the ATO semiconducting particles dispersed in water. 2.5% (w/v) ATO was added to a working electrode reagent containing an enzyme and a mediator and other reagent components (buffer, surfactant, etc.).

[00135] FIG. 8 is a graph illustrating the effect that the inclusion of semiconducting particles in the sensor has on the active working electrode area. Glucose test strips were fabricated from FreeStyle test strip halves consisting of carbon working electrodes and Ag/AgCl electrodes in a facing configuration. The working electrode included surfactant, buffer, MSG and ATO nanoparticles, where present. Counter reference electrodes included first and second surfactants, NaCl and a buffer.

[00136] Note that all enzyme and mediator were omitted from the carbon electrode half, and therefore the "blank" test strip became a convenient platform for studying the

electrochemical behavior of the ATO alone. The above strip halves were assembled to construct a thin-layer electrochemical cell, the strip was filled with 0.1M phosphate buffer saline (PBS), pH 7, and cyclic voltammetry was performed over the range -0.8V to +0.8V, at a scan rate of 50 mV/second (the potential of the carbon-containing strip half was scanned relative to that of the Ag/AgCl containing half). The double layer charging current was evaluated from the resulting curve, using the current difference between anodic and cathodic scans at 0V. As shown in FIG. 8, the two strips with the ATO semiconducting particles (labeled "ATO strip" and "Sensor strip") showed a significant increase in double layer charging (ca. 10 μΑ) compared with the ATO-free strip ("Blank strip" double layer charging current less than 1 μΑ). This change in double layer charging current suggests the added ATO does not act simply as in inert filler material, but also acts as an extension of the electrode, increasing the total electroactive area.

[00137] Another experiment compared the peak currents of strips fabricated with no added particles (control), inert particles, conducting particles (carbon nanotubes), and semiconducting particles (ATO). The strips were fabricated as above in FIG. 8, except all strips contained in addition 23.7 units/cm2 of enzyme and 9 mg/cm2 of mediator. Further, semiconductor particles were replaced with conducting or inert particles, as appropriate:

[00138] The strips with ATO semiconducting particles exhibited a peak current of 99.3 μΑ, intermediate between that of the control (84.8 μΑ), and the conducting particles (109.7 μΑ). Importantly, this value is substantially increased as compared to a strip with inert particles (virtually unchanged from control). High peak current is indicative of both well distributed chemistry and an increased electroactive area. This provides evidence that semiconducting particles increase electrode area and performance relative to both control and inert particles, while avoiding the short circuits possible with conducting particles.

[00139] FIG. 9 and FIG. 10 compare the fill times and reproducibility of strips incorporating ATO semiconducting particles with sensors that do not include the semiconducting particles. Three strip formulations were studied that had different reagent material without semiconducting particles (A, B and C) and two strip formulations were studied that included ATO semiconducting particles in the reagent solution (D and E). The designations A, B, C refer to three versions of enzyme. The D and E strips contained equal amounts of ATO

semiconductor particles and reduced enzyme A loading, with sample D having 50% of the loading as sample A and sample E having 75% of the loading of sample A. Counter-reference formulations were the same as FIG. 8.

[00140] As shown in FIG. 9, after eight weeks at 56°C and 65°C, the fill time of the sensors D and E with ATO semiconducting particles showed little change in fill time while the sensors A, B, C without the semiconducting particles showed significant increases in fill time. Fill time is a sensitive indicator of strip degradation, and the results indicate that the addition of ATO has significantly stabilized the strips. [00141] Similarly, FIG. 10 illustrates the increased reproducibility of the strips incorporating ATO semiconducting particles compared to strips that did not incorporate the semiconducting particles. The reproducibility is measured as a coefficient of variation (CV) for the glucose test result, after eight weeks at 25°C, 56°C and 65°C. After eight weeks at each of the temperatures, the CV of the strips D and E with ATO semiconducting particles remained low and nearly constant, while the sensors A, B, C without the semiconducting particles had higher CVs, particularly at 56°C and 65°C.

[00142] FIG. 11A illustrates the improved bias control when ATO semiconducting particles are incorporated into the strip reagent. A sample was prepared using a particular enzyme and carbon, with a second sample using the same enzyme and carbon prepared with ATO particles. The samples were tested at low, middle and high glucose levels. As shown in FIG. 11 A, bias control was improved at higher glucose levels when ATO semiconducting particles were incorporated.

[00143] The same samples were tested for fill time of the sensors, as represented in FIG.

1 IB. Consistent with the results shown in FIG. 9, the fill time for sensors incorporating ATO semiconducting particles was improved at both 25°C and 56°C.

[00144] FIGs. 12A and 12B illustrate the charge results for strips with and without semiconducting particles at various levels of reagent (enzyme and mediator) loading tested at various glucose levels. Reagent loadings were varied from 40% to 100%. The counter-reference electrode formulations are unchanged from FIG. 8.

[00145] As shown in FIG. 12A, some non-linearity is observed in the control strips (no

ATO) at all reagent loading levels. The non-linearity is a product of the uneven reagent deposition on the working electrode when no semiconducting particles are used. As reagent loading is decreased to 40%, this non-linearity increases substantially. The maximum charge decreased from 700 microcoulombs at 100% reagent loading to 340 microcoulombs at 40% reagent loading, both measurements taken at 350 mg/dL glucose.

[00146] FIG. 12B illustrates the charge results for strips with the ATO semiconducting particles at various levels of reagent loading tested at various glucose levels. The ATO level was not changed. Reagent loadings were varied from 40% to 100% at varying levels of glucose as in FIG. 12A. As shown in FIG. 1 IB, linearity is nearly identical for all strips, irrelevant of the reagent loading. In addition, the maximum charge only varies between 750 microcoulombs at 60% reagent loading and 800 microcoulombs at 40% reagent loading, both measurements taken at 350 mg/dL glucose. The results in FIG. 1 IB indicate superior recovery at the various loadings for the strips with ATO semiconducting particles as compared with the strip without

semiconducting particles at 100% loading.

[00147] The results shown in FIG. 12B also indicate that reagent loading can be reduced with the use of ATO semiconducting particles.

[00148] To illustrate uniform reagent distribution after coating, a head-to-head coating study was performed. The coating quality of sensors with and without ATO semiconducting particles added to the reagent solution was evaluated. The results were presented as a solution fail rate. Two printed carbon rolls were used in the study. The solution fail rate was 6.4% for the sensors without the ATO semiconducting particles, while the solution fail rate was 1.5% with the ATO semiconducting particles.

[00149] The precision of sensors having the same reagent formulation using the same glucose levels was tested, with one set of sensors without ATO and one set of sensors with ATO. FIGs. 13A and 13B illustrate the results of the tests, with FIG. 13A illustrating the precision of the sensor formulation without ATO and FIG. 13B illustrating the precision of the sensor formulation with ATO. As illustrated, the sensors using the ATO nanoparticle formulation demonstrated better precision than the sensors having the non-ATO nanoparticles formulation.

[00150] The foregoing results indicate that sensors using the ATO nanoparticle

formulation have advantages in minimizing the performance impact from unknown carbon ink variation, printing variation and chemistry coating variation, resulting in better strip precision, better sample filling performance and potentially better yield.

[00151] Although the foregoing invention has been described in some detail by way of illustration and example for purposes of clarity of understanding, it is readily apparent to those of ordinary skill in the art in light of the teachings of this invention that certain changes and modifications may be made thereto without departing from the spirit or scope of the appended claims. Accordingly, the preceding merely illustrates the principles of the invention.

[00152] It will be appreciated that those skilled in the art will be able to devise various arrangements which, although not explicitly described or shown herein, embody the principles of the invention and are included within its spirit and scope. Furthermore, all examples and conditional language recited herein are principally intended to aid the reader in understanding the principles of the invention and the concepts contributed by the inventors to furthering the art, and are to be construed as being without limitation to such specifically recited examples and conditions. Moreover, all statements herein reciting principles, aspects, and embodiments of the invention as well as specific examples thereof, are intended to encompass both structural and functional equivalents thereof. Additionally, it is intended that such equivalents include both currently known equivalents and equivalents developed in the future, i.e., any elements developed that perform the same function, regardless of structure. The scope of the present invention, therefore, is not intended to be limited to the exemplary embodiments shown and described herein. Rather, the scope and spirit of present invention is embodied by the appended claims.

Claims

What is claimed is:

1. A sensor for determining the concentration of an analyte in a sample fluid, the sensor comprising:

a first substrate having a proximal end and a distal end, the first substrate defining a first side edge and a second side edge of the sensor extending from the proximal end to the distal end of the first substrate, the distal end being configured and arranged for insertion into a sensor reader;

a second substrate disposed over the first substrate;

a working electrode disposed on one of the first and second substrates;

a counter electrode disposed on one of the first and second substrates;

a spacer disposed between the first and second substrates and defining a sample chamber that comprises the working electrode and the counter electrode;

an analyte detection reagent disposed on the working electrode; and

semiconducting particles disposed on at least a portion of a surface of the sample chamber.

2. The sensor of claim 1, wherein the semiconducting particles comprise antimony doped tin oxide.

3. The sensor of claim 1, wherein the analyte detection reagent and the semiconducting particles form a composition, the composition disposed on an area of the working electrode, the semiconducting particles configured and arranged to provide for substantially uniform distribution of the detection reagent on the area of the working electrode.

4. The sensor of claim 3, wherein the area of the working electrode on which the composition is disposed comprises between about 0.001 and 10 mg of semiconducting particles per cm².

5. The sensor of claim 1, wherein the analyte detection reagent and the semiconducting particles form a composition, the composition disposed on an area of the working electrode, the semiconducting particles configured and arranged to increase an effective surface area of the working electrode.

6. The sensor of claim 5, wherein the semiconducting particles increase the effective surface area of the working electrode by between about 50 and 1000 percent.

7. The sensor of claim 1, wherein the semiconducting particles are disposed on the first substrate in the sample chamber and configured to improve sample fill time consistency.

8. The sensor of claim 1, wherein the semiconducting particles are disposed on the second substrate in the sample chamber and configured to improve sample fill time consistency.

9. The sensor of claim 1, wherein the semiconducting particles are disposed on at least one surface of the spacer in the sample chamber and configured to improve sample fill time consistency.

10. The sensor of any one of claims 1 to 9, wherein the semiconducting particles are disposed as substantially a monolayer on the area of the working electrode.

11. The sensor of any one of claims 1 to 9, wherein the semiconducting particles are disposed as substantially a bilayer on the area of the working electrode.

12. The sensor of any one of claims 1 to 9, wherein the semiconducting particles are disposed as more than two layers on the area of the working electrode.

13. The sensor of claim 1, wherein the working electrode and counter electrode independently comprise a material selected from the group consisting of: gold, carbon, platinum, ruthenium, palladium, silver, silver chloride, silver bromide, and combinations thereof.

14. The sensor of claim 1, wherein the working electrode is disposed on the first substrate and the counter electrode is disposed on the second substrate.

15. The sensor of claim I, wherein the working electrode and the counter electrode are disposed on the same substrate.

16. The sensor of claim 1, wherein the sample chamber is sized to contain a volume of no more than about 1 μΐ, of sample fluid.

17. A method for determining the concentration of an analyte in a sample, comprising the steps of:

contacting a sample with a sensor, the sensor comprising:

a second substrate disposed over the first substrate;

a working electrode disposed on one of the first and second substrates;

a counter electrode disposed on one of the first and second substrates;

an analyte detection reagent disposed on the working electrode; and a semiconducting particles disposed on at least a portion of a surface area of the sample chamber;

generating a sensor signal at the working electrode; and

determining the concentration of the analyte using the sensor signal.