Compensating for inadequate sample volume in biosensor devices

CA 02838176 2015-09-03

COMPENSATING FOR INADEQUATE SAMPLE VOLUME

IN BIOSENSOR DEVICES

This patent application is a divisional patent application of Canadian patent application 2,407,249 filed October 9, 2002.

Field Of The Invention

The field of this invention is the electrochemical determination of analyte in biological fluids, particularly the electrochemical determination of the adequacy of the volume of the biological fluid sample to be tested for analyte concentration.

Background Of The Invention

Analyte concentration determination in biological fluids, e.g., blood or bloodderived products such as plasma, is of ever increasing importance to today’s society. Such assays find use in a variety of applications and settings, including clinical laboratory testing, home testing, etc., where the results of such testing play a prominent role in the diagnosis and management of a variety of disease conditions. Common analytes of interest include glucose for diabetes management, cholesterol for monitoring cardiovascular conditions, and the like. In response to this growing importance of analyte concentration detection, a variety of analyte detection protocols and devices for both clinical and home use have been developed.

One type of method that is employed for analyte detection is an electrochemicalbased method. In such methods, an aqueous liquid sample is placed into a reaction zone in an electrochemical cell made up of at least two electrodes, i.e., a counter/reference electrode and a working electrode, where the electrodes have an impedance which renders them suitable for amperometric measurement. The component to be analyzed, z.e., analyte, is allowed to react directly with an electrode, or directly or indirectly with a redox reagent to form an oxidisable (or reducible) substance in an amount corresponding to the concentration of the analyte. The quantity of the oxidisable (or reducible) substance present is then estimated electrochemically and related to the amount of analyte present in the initial sample.

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Commonly, the electrochemical cell is in the form of a disposable test strip on which the biological sample is deposited and which is receivable within a meter by which the electrochemical analyte concentration measurement is made. Examples of assay systems that employ these types of test strips, often referred to as biosensors, and meters may be found in U.S. Patent Nos. 5,942,102, 6,174,420 Bl and 6,179,979 Bl. With these systems, determination of the concentration of an analyte in a biological sample first involves obtaining a biological sample and bringing that sample into contact with a reaction area of the test strip so that the biological sample, and more particularly the analyte of interest or derivative thereof, may react with the chemistry, e.g., the testing reagent(s), associated with the reaction area. In order to obtain an accurate measurement of the particular analyte(s) of interest, a minimum sample volume must be applied to the reaction area. It is not uncommon for an inadequate amount of sample volume to be provided, often due to user error or patient inexperience or misjudgment. Inaccurate measurements can result in a misdiagnosis or improper treatment, such as administering an inappropriate dosage of a drug, patient non-compliance, etc. Such can result in serious and even life-threatening consequences for those whose lives depend on frequent monitoring of an analyte in their body, for example, diabetics.

One approach to ensuring an adequate biological sample volume is to over-saturate or use a greater volume of sampled fluid than is necessary to fill the reaction area of the test strip. A disadvantage of using an unnecessarily large volume of a sampled fluid, a blood sample in particular, is the need to draw a greater volume of blood sample from the patient. This requires use of a blood sample volume, which is rather large, thus necessitating use of a larger diameter needle and/or deeper penetration into the skin. These factors can increase the discomfort and pain felt by the patient, and may be difficult to achieve for those individuals whose capillary blood does not readily express. As this sampling process may be repeated frequently within a single day, for many diabetics, for example, an increase in pain quickly becomes less tolerable or intolerable all together.

Some analyte detection biosensors have been developed to provide visual confirmation of the adequacy of sample volume, however, this feature does not exclude potential error by the patient in judging the adequacy of the sample’s volume, e.g., diabetics may experience deteriorated vision. Certain other analyte determination biosensors do provide user-independent means for determining the adequacy of the sample volume.

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Examples of such biosensors are disclosed in U.S. Patent Nos. 5,628,890 and 5,650,062 and PCT Patent Application Publication No. WO 99/32881 (PCT Patent Application No. PCT/US98/27203). In particular, the ‘881 publication describes an electrochemical glucose monitoring system which attempts to determine the adequacy of a volume of sample applied to a biosensor by applying a low-level AC voltage signal (without a DC voltage offset) at a known frequency to the biosensor and then measuring both the real component and the imaginary component of the resulting impedance. These impedance values are then compared to a look-up table in the microprocessor’s program memory. The accuracy of this method may be additionally questionable considering that this system is dependent on blood hematocrit levels and environmental temperature variations.

Another disadvantage of the technique disclosed in the ‘881 publication is that the analyte measurement test must be aborted if the sample volume is determined to be inadequate, i.e., a “go-no-go” situation. This results in the need to take yet another sample from the patient which, as mentioned above, is inconvenient and may be very painful to the patient, likely resulting in patient non-compliance in his or her medication regime. Additionally, the test must be repeated resulting in the waste of test strips and increasing the cost of the procedure.

As such, there is continued interest in the identification of new techniques for accurately and precisely measuring the adequacy of the volume of the sample used for electrochemical analyte concentration determination. Of particular interest would be the development of methods that can very accurately and expeditiously determine the adequacy of the volume of sample. It would be additionally beneficial to develop such a sample volume adequacy determination technique in which a determination that a sample volume is inadequate does not require abortion of the analyte concentration measurement test. Ideally, this technique would compensate for the less than optimal sample volume and provide an accurate analyte concentration measurement without having to provide a new sample or conduct a new test.

Summary of the Invention

This disclosure provides methods and systems for measuring the volume of

CA 02838176 2013-12-23 biological sample and determining whether such volume is adequate to produce an accurate measurement of at least one selected characteristic of the biological sample, such as the concentration of an analyte contained therein. Certain such methods and systems provide the additional function of compensating for a sample volume determined to be less than adequate in order to proceed with a measurement procedure.

The present invention is employed with a biosensor, such as an electrochemical test strip to which the sample volume of biological solution is deposited, and a meter configured to receive such test strip and to measure the concentration of selected analytes within the biological sample. The electrochemical test strip, as will be more fully described below, includes an electrochemical cell comprised of opposing electrodes between which a reaction zone is defined for receiving the biological sample, wherein the reaction zone has a defined thickness and volume.

When sufficient voltage is applied to an electrochemical cell, both double-layer charging and electrochemical reaction will occur. As a consequence, charge flows to the electrodes of an electrical cell. The electrode-solution interface is analogous to that of a capacitor. The ratio of this charge to the voltage determines the capacitance of the electrodesolution interface. Since the total charge is due to the charging of the double layer and to the electrochemical reaction, two distinct capacitance components, Cji and C_s, respectively, contribute to the total or equivalent capacitance of the cell (see Bard, A.J. and Faulkner, L.R., Electrochemical Methods, 1980).

The inventors have discovered that the equivalent capacitance of an electrochemical cell is the most relevant factor in precisely determining sample volume, as the equivalent cell capacitance is linearly proportional to the amount of surface area of the cell electrodes in contact with the sample (the “covered cell area”), and thus, is linearly proportional to the volume of the sample within the cell, i.e., between the electrodes. The inventors have also discovered that the equivalent resistance of the electrochemical cell is additionally relevant in precisely determining sample volume, as the equivalent cell resistance is inversely proportional to the covered cell area, and thus, is inversely proportional to the sample volume.

Thus, a feature disclosed is to derive such covered cell area and the corresponding

CA 02838176 2013-12-23 sample volume from the equivalent cell capacitance or from both the equivalent cell capacitance and the equivalent cell resistance.

Another feature disclosed is to control certain other factors (e.g., the thickness of the cell, the concentration of ionic species, etc.) that may interfere with accurately measuring sample volume in order that the value of the equivalent cell capacitance is independent and unaffected by the glucose concentration and blood hematocrit levels within the sample, the environmental temperature, particularities of the blood donor and other commonly interfering components of blood.

Yet another feature of the present invention is to provide the additional function of compensating for a sample volume determined to be less than adequate in order to proceed with an accurate analyte concentration measurement.

Accordingly, there is provided methods for determining the adequacy of the volume of a biological sample to be used for determining the concentration of one or more selected analytes within the donor sample, which achieves these objectives and provides these features.

In certain embodiments of the subject methods, an alternating current voltage (AC voltage) of low amplitude and having a selected frequency is applied to a biosensor containing the biological sample to be tested, thereby charging the biosensor. Optionally, a direct current voltage (DC voltage) may be applied simultaneously along with the AC voltage in order to increase the rate at which the capacitance of the biosensor becomes stabilized. The resulting alternating current generated from such charging is then measured, and the equivalent cell capacitance of the biosensor is then determined from the resulting alternating current. The equivalent cell capacitance is then used to determine the amount of surface area of the biosensor in contact with the sample solution, which surface area is then used to derive the volume of the sample within the biosensor. Upon a determination that the sample volume is adequate to make an accurate analyte concentration measurement, such analyte concentration is measured. On the other hand, if it is determined that the sample volume is inadequate, the subject methods may further include compensating for such inadequate sample volume during the analyte concentration measurement process. Inadequate volume compensation involves determining the necessary compensation factor

CA 02838176 2015-09-03 which includes, at least in part, determining the ratio of the equivalent cell capacitance of the biosensor containing the actual sample volume to the cell capacitance of the biosensor when its entire available volume is filled.

Also provided are systems for carrying out the subject methods. The subject systems include electronic components and/or circuitry intended to be used with and electronically coupled to a biosensor, e.g., an electrochemical measurement cell in the form of, e.g., a disposable test strip, into which the sampled solution to be tested is deposited or is drawn by a capillary action. Most typically, such electronic circuitry is incorporated into a meter or other automated device configured to receive and operatively engage with such electrochemical cell, e.g., a disposable test strip, and to measure one or more physical or chemical characteristics of a biological sample held within the electrochemical cell. Most typically, such characteristics include the concentration of one or more target analytes within the biological sample. Such electronic circuitry may comprise discrete electronic components, e.g., a voltage supply, and/or integrated circuits having multiple circuit elements and/or semiconductor devices, e.g., a microprocessor suitably programmed to execute certain steps or functions of the subject methods based on certain signal or data inputs received from the electrochemical cell.

In certain embodiments, the systems of the present invention include such electronic circuitry and such an automated measurement device or meter, as just described, wherein the electronic circuitry is completely structurally and functionally integral with the automated measurement device.

While the subject methods and systems may be used to determine the sample volume of different biological samples, such as urine, tears, saliva, and the like, they are particularly suited for use in determining the sample volume of blood or blood fractions and the like. Furthermore, while the subject systems and methods for determining the sample volume in preparation for measuring a variety of physical and chemical characteristics of the sample, they are particularly useful in preparation for measuring the concentration of selected analytes within the sample.

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In one embodiment, there is provided a method for compensating for an inadequate volume of a biological sample held within an electrochemical biosensor for measurement of at least one characteristic of the biological sample. The method includes: determining the volume of the biological sample; determining whether the volume of the biological sample is adequate; and compensating for an inadequate volume of the biological sample held within the electrochemical biosensor based on a determination that the volume is inadequate, wherein determining a necessary compensation factor comprises determining a ratio of the capacitance of the biosensor when completely filled with the sample volume to the capacitance of the biosensor with the inadequate sample volume.

In one embodiment, there is provided a method for measuring at least one characteristic of a biological sample held within an electrochemical biosensor. The method includes: determining the volume of the biological sample held within the electrochemical biosensor using a capacitance of the biosensor; determining whether the volume of the biological sample is adequate for measuring the at least one characteristic; and upon determining that the volume of the biological sample is inadequate measuring the at least one characteristic of the biological sample and, compensating the at least one characteristic measurement for the inadequate volume, wherein compensating for an inadequate volume comprises determining a ratio of the capacitance of the biosensor when completely filled with the sample volume to the capacitance of the biosensor with the inadequate sample volume.

In one embodiment, there is provided a system for compensating for an inadequate volume of a biological sample held within an electromagnetic biosensor for measurement of at least one characteristic of the biological sample. The system includes: a voltage supply configured for applying a voltage to an electrochemical cell of the biosensor; means for measuring a current generated by the cell when the cell voltage is applied to the cell; means for deriving a capacitance of the cell from the measured current; means for deriving a surface area of the cell covered by the biological sample from the cell capacitance; means for deriving the volume of the biological sample from the cell surface area; means for determining whether the sample volume is adequate for making an accurate measurement of the concentration of one or more selected analytes within the biological sample; means for

6a

CA 02838176 2015-09-03 measuring the concentration of one or more selected analytes within the biological sample; and means for compensating for the sample volume determined to be inadequate for measuring the concentration of one or more selected analytes within the biological sample, wherein the compensation for inadequate volume is based on a ratio of the capacitance of the cell when completely filled to the capacitance of the cell with the inadequate sample volume.

In one embodiment, there is provided a system for use with a meter configured for engaging an electrochemical cell and measuring the concentration of one or more selected analytes within a biological sample held within the electrochemical cell. The system includes: a voltage supply configured for applying an alternating and/or a direct current voltage to the cell; and an electronic circuit configured for receiving a current generated by the electrochemical cell when a voltage is applied to the cell, measuring the generated current, determining a capacitance of the cell from the measured current, determining a surface area of the cell covered by the biological sample from the cell capacitance, and determining the volume of the biological sample from the cell surface area covered by the biological sample, and wherein the electronic circuit is further configured for determining whether the sample volume is adequate for making an accurate measurement of one or more selected physical or chemical characteristics of the biological sample; wherein the electronic circuit is further configured for compensating for the sample volume if it is determined to be inadequate for measuring one or more selected physical or chemical characteristics of the biological sample.

In another embodiment, there is provided a system for measuring selected characteristics of a biological sample held within a test strip. The system includes: a meter configured to receive the test strip and comprising a voltage supply configured for applying an alternating and/or a direct voltage to the test strip; and a microprocessor electronically coupled to the meter and comprising: (i) means for storing data related to the test strip, the biological sample, calibration and performance parameters of the meter; (ii) means for receiving a cunent generated by the electrochemical test strip when a voltage is applied to the test strip; (iii) means for measuring the generated current; (iv) means for determining the volume of the biological sample from a test strip surface area covered by the biological

6b

CA 02838176 2015-09-03 sample based on the measured current; and (v) means for measuring the selected characteristics; wherein the microprocessor further comprises means for determining whether the sample volume is adequate for measuring the selected characteristics and means for compensating a selected characteristic measurement for an inadequate sample volume, wherein compensation for inadequate volume is based on a ratio of the capacitance of the test strip when completely filled to the capacitance of the test strip with the inadequate sample volume.

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 methods and

6c

CA 02838176 2013-12-23 systems of the present invention which are more fully described below.

Brief Description of the Drawings

Fig. 1 is an exploded view of an exemplary conventional electrochemical test strip for electrochemical analyte concentration determination, which is usable with the present invention.

Fig. 2 is a schematic illustration of a circuit representative of the equivalent cell impedance of the test strip of Fig. 1.

Fig. 3 is a block diagram illustration of a system of the present invention operatively coupled to an electrochemical biosensor for measuring the equivalent cell capacitance of the electrochemical biosensor when a voltage is applied to the biosensor.

Fig. 4 is a graph depicting the relationship of the change in the equivalent cell capacitance (y-axis) over time (x-axis) of the electrochemical cell of the test strip of Fig. 1 when the cell is completely filled and half filled, respectively, with a sampled solution.

Fig. 5 is a graph depicting the relationship of the change in the equivalent resistance (y-axis) of the electrochemical over time (x-axis) when the cell of the test strip of Fig. 1 is completely filled and half filled, respectively, with a sampled solution.

Fig. 6 is a histogram depicting the relationship of the change in the equivalent cell capacitance (x-axis) of the electrochemical cell of the test strip of Fig. 1 at 0.5 seconds after application of the sample solution to the test strip (y-axis) when the cell is completely filled and half filled, respectively, with a sampled solution.

Fig. 7 is a graph depicting the ratio of the equivalent cell capacitance and the equivalent cell resistance (y-axis) over time (x-axis) when the electrochemical cell of the test strip of Fig. 1 is completely filled and half filled, respectively, with a sampled solution.

Fig. 8 is a scatter diagram of the ratio of the equivalent cell capacitance and the equivalent cell resistance (y-axis) versus the equivalent cell capacitance (x-axis) measured at 0.5 seconds following sample application when the electrochemical cell is completely filled and half filled, respectively, with a sampled solution.

CA 02838176 2015-09-03

Detailed Description of the Preferred Embodiments

The present invention provides systems and methods for determining the volume of a biological sample for purposes of measuring a selected characteristic of the sample, e.g., analyte concentration, and determining whether such volume is adequate to produce an accurate measurement of such selected characteristic. Certain such systems and methods provide the additional function of compensating for a sample volume determined to be less than adequate in order to provide an accurate measurement of such characteristic.

Before the present invention is described in further detail, it is to be understood that this invention is not limited to the particular embodiments described, as such may, of course, vary. It is also to be understood that the terminology used herein is for the purpose of describing particular embodiments only.

Where a range of values is provided, it is understood that each intervening value, to the tenth of the unit of the lower limit unless the context clearly dictates otherwise, between the upper and lower limit of that range and any other stated or intervening value in that stated range is encompassed within the invention. The upper and lower limits of these smaller ranges may independently be included in the smaller ranges is also encompassed within the invention, subject to any specifically excluded limit in the stated range. Where the stated range includes one or both of the limits, ranges excluding either both of those included limits are also included in the invention.

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 this invention belongs. Although any methods and materials similar or equivalent to those described herein can also be used in the practice or testing of the present invention, a limited number of the exemplary methods and materials are described herein.

It must be 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.

The publications discussed herein are provided solely for their disclosure prior to the filing date of the present application. Nothing herein is to be construed as an admission that the present invention is not entitled to antedate such publication by virtue of prior invention.

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Further, the dates of publications provided may differ from the actual publication dates, which may need to be independently confirmed.

Definitions

The term “double layer” as used herein refers to the whole array of charged species and oriented dipoles existing at the interface between an electrode surface and a solution, e.g., a sample of a biological solution, in contact with the electrode surface when a voltage is applied to the electrode.

The term “double layer capacitance,” Cdi, as used herein means the capacitance contributed by the charging of the double layer of the electrode-solution interface.

The term “Faradaic capacitance,” C_s, as used herein refers to the pseudocapacitance component due to the electrochemical reaction process that occurs on the electrode surface.

The term “Faradic current,” ip, as used herein means the current or electron transfer that occurs at the surface of an electrode to which a voltage has been applied. The term “equivalent cell capacitance,” C, when used herein in reference to an electrochemical cell means the total equivalent capacitance across the electrochemical cell, which results when a voltage has been applied to the electrochemical cell. The equivalent cell capacitance is dominated by the double layer capacitance and the Faradaic capacitance .

The term “equivalent cell resistance,” R, as used herein in reference to an electrochemical cell means the total equivalent resistance across the electrochemical cell, which results when a voltage has been applied to electrochemical cell.

The “equivalent cell impedance,” Z, as used interchangeably herein in reference to an electronic circuit or component, e.g., an electrochemical cell, means the total impedance of the circuit including but not necessarily limited to the combination of the equivalent cell capacitance and the equivalent cell resistance, which results when a voltage has been applied to the electrochemical cell.

The term “Faradaic current,” ip, as used herein, means the current resulting from electron transfer between sample component and surface of an electrode as a result of electrochemical reaction when a voltage has been applied.

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The terms “derive,” “determine,” “calculate,” and the like, and their respective derivatives, are used interchangeably herein.

The present invention will now be described in detail. In further describing the present invention, exemplary electrochemical biosensors, usable with the systems and employable by the methods of the present invention, will be described first, followed by a detailed description of the subject methods and systems, as well as a description of the subject kits that include the subject systems for use in practicing the subject methods.

Electrochemical Biosensors

As summarized above, the invention provides methods and systems for measuring the volume of a sample of biological material used for analyte concentration measurement and determining whether such volume is adequate to produce an accurate analyte concentration measurement. These methods and systems are usable with a biosensor, more particularly an electrochemical cell-based biosensor, into which the sampled biological material is deposited or transferred. There are varying designs of electrochemical cell-based biosensors. The most common of these designs employed in the field of analyte concentration monitoring include test strip configurations, such as those disclosed in U.S. Patent Nos. 6,193,873; 6,475,372; 6,716,577; 6,620,310 and 6,558,528.

In U.S. 6,193,873, an applied sample bridges a gap between electrodes. A constant small current is applied across the gap and the time the sample bridges the gap is marked by a sharp drop in potential. A constant voltage is applied after the sample is detected, and the cunent and/or charge through the sample is monitored over a period of time. From the measured current or charge, the analyte concentration of interest is calculated. In one taught embodiment, the substrate is a polyester base on which has been deposited - typically, by sputtering - a Pd coating, which forms the working electrode. A dry reagent, consisting of buffer, mediator, and enzyme, is deposited near one end of the electrode. The spacing layer is a double-sided adhesive having a cutout that defines the electrochemical cell. Typically the spacer is less than about 200 pm thick. A top layer is a polyester layer on which has been deposited - typically by sputtering, also - a Au coating, which forms the reference electrode. A device of this type can use a glucose oxidase (GOD)/ferricyanide system to determine glucose concentrations.

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In U.S. 6,475, 372, the physiological sample is introduced into an electrochemical cell having a working and reference electrode. A first electric potential is applied to the cell and the resultant cell current over a period of time is measured to determine a first timecurrent transient. A second electric potential of opposite polarity is then applied and a second a time-current transient is determined. The preliminary concentration of the analyte is then calculated from the first and/or second time-current transient and this preliminary analyte concentration less a background value is then multiplied by a hematocrit correction factor to obtain the analyte concentration in the sample. The electrochemical test strips are made up of two opposing metal electrodes separated by a thin spacer layer, where these components define a reaction area or zone in which is located a redox reagent system. The working and reference electrodes may be generally configured in the form of elongated rectangular strips with length ranges from about 1.9 to 4.5 cm, usually from about 2.0 to 2.8 cm and width ranges from about 0.38 to 0.76 cm, usually from about 0.51 to 0.67 cm. The reference electrodes typically have a thickness ranging from about 10 to 100 nm and usually from about 10 to 20 nm. In certain embodiments, the length of one of the electrodes is shorter than the length of the other electrode, typically about 0.32 cm. The shorter electrode may be the working or reference electrode. At least the surface of the working and reference electrodes that faces the reaction area in the strip is a metal, where metals of interest include palladium, gold, platinum, silver, iridium, carbon, doped tin oxide, stainless steel and the like. While in principle the entire electrode may be made of the metal, each of the electrodes is generally made up of an inert support material on the surface of which is present a thin layer of the metal component of the electrode. The thickness of the inert backing material typically ranges from about 51 to 356 gm, usually from about 102 to 153 pm while the thickness of the metal layer typically ranges from about 10 to 100 nm and usually from about 10 to 40 nm, e.g. a sputtered metal layer. U.S. 6,475,372 teaches that any convenient inert backing material may be employed in the electrodes, where typically the material is a rigid material that is capable of providing structural support to the electrode and, in turn, the electrochemical test strip as a whole. Suitable materials that may be employed as the backing substrate include plastics, e.g. PET, PETG, polyimide, polycarbonate, polystyrene, silicon, ceramic, glass, and the like. The working and reference electrodes face each other and are separated by only a short distance. This minimal spacing of the working and reference electrodes in the test strips is a result of the presence of a thin spacer layer positioned, or sandwiched between the working and reference electrodes. The thickness of this spacer layer

CA 02838176 2013-12-23 generally should be less than or equal to 500 μηι, and usually ranges from about 102 to 153 gm. The spacer layer is cut so as to provide a reaction zone or area with at least an inlet port into the reaction zone, and generally an outlet port out of the reaction zone as well. The spacer layer may have a circular reaction area cut with side inlet and outlet vents or ports, or other configurations, e.g. square, triangular, rectangular, irregular shaped reaction areas, etc. The spacer layer may be fabricated from any convenient material, where representative suitable materials include PET, PETG, polyimide, polycarbonate, and the like, where the surfaces of the spacer layer may be treated so as to be adhesive with respect to their respective electrodes and thereby maintain the structure of the electrochemical test strip. Of particular interest is the use of a die-cut double-sided adhesive strip as the spacer layer. The working and reference electrodes define the top and bottom of the reaction area, while the spacer layer defines the walls of the reaction area. The volume of the reaction area is at least about 0.1 gL, usually at least about 1 pL and more usually at least about 1.5 pL, where the volume may be as large as 10 pL or larger. The reaction area generally includes at least an inlet port, and in many embodiments also includes an outlet port. The cross-sectional area of the inlet and outlet ports may vary as long as it is sufficiently large to provide an effective entrance or exit of fluid from the reaction area, but generally ranges from about 9x10‘⁴ to 5x10‘³ cm², usually from about 1.3x1 O'³ to 2.5x10’³ cm². Present in the reaction area is a redox reagent system, which reagent system provides for the species that is measured by the electrode and therefore is used to derive the concentration of analyte in a physiological sample. The redox reagent system present in the reaction area typically includes at least an enzyme(s) and a mediator. In many embodiments, the enzyme member(s) of the redox reagent system is an enzyme or plurality of enzymes that work in concert to oxidize the analyte of interest. Enzymes of interest include oxidases, dehydrogenases, lipases, kinases, diphorases, quinoproteins, and the like. The specific enzyme present in the reaction area depends on the particular analyte for which the electrochemical test strip is designed to detect, where representative enzymes include: glucose oxidase, glucose dehydrogenase, cholesterol esterase, cholesterol oxidase, lipoprotein lipase, glycerol kinase, glycerol-3phosphate oxidase, lactate oxidase, lactate dehydrogenase, pyruvate oxidase, alcohol oxidase, bilirubin oxidase, uricase, and the like. Where the analyte of interest is glucose, the enzyme component of the redox reagent system may be a glucose oxidizing enzyme, e.g. a glucose oxidase or glucose dehydrogenase. The second component of the redox reagent system is a mediator component, which is made up of one or more mediator agents. A

CA 02838176 2013-12-23 variety of different mediator agents are known in the art and include: ferricyanide, phenazine ethosulphate, phenazine methosulfate, pheylenediamine, 1-methoxy-phenazine methosulfate,

2,6-dimethyl-l,4-benzoquinone, 2,5-dichloro-l,4-benzoquinone, ferrocene derivatives, osmium bipyridyl complexes, ruthenium complexes, and the like. Where glucose in the analyte of interest and glucose oxidase or glucose dehydrogenase are the enzyme components, mediators of particular interest are ferricyanide, and the like. Other reagents that may be present in the reaction area include buffering agents, e.g. citraconate, citrate, malic, maleic, phosphate, Good buffers and the like. Yet other agents that may be present include: divalent cations such as calcium chloride, and magnesium chloride; pyrrolbquinoline quinone; types of surfactants such as Triton, Macol, Tetronic, Silwet, Zonyl, and Pluronic; stabilizing agents such as albumin, sucrose, trehalose, mannitol, and lactose. The redox reagent system is generally present in dry form. The amounts of the various components may vary, where the amount of enzyme component typically ranges from about 1 to 100 mg/mL, usually from about 5 to 80 mg/mL; and the amount of mediator component typically ranges from about 5 to 1000 mM, usually from about 90 to 900 mM.

In U.S. 6,716, 577, the test strips have a reaction zone defined by opposing metal electrodes separated by a thin spacer layer. The metal surface of at least one of the electrodes is modified by a homogenous surface modification layer made up of linear self-assembling molecules having a first sulfhydryl end group and a second sulfonate end group separated by a short chain alkyl linking group, 2-mercaptoethane sulfonic acid or a salt thereof being preferred in certain embodiments. For the shape, dimensions, metals and inert backing material taught, reference can be had generally to the teachings above for US 6,475,372, with the reference electrodes typically having a thickness ranging from about 10 to 100 nm and usually from about 18 to 22 nm and the thickness of the inert backing material typically ranging from about 51 to 356 pm, usually from about 10 to 153 pm while the thickness of the metal layer typically ranges from about 10 to 100 nm and usually from about 20 to 40 nm, e.g. a sputtered metal layer. At least one of the metallic surfaces of the electrodes, and in some embodiments both of the metallic surfaces of the electrodes, that face, i.e. border or bound, the reaction area, have a surface modification layer present thereon. The surface modification layer is a homogenous layer of self-assembling molecules that renders the surface stably hydrophilic in a storage stable manner. More specifically, the surface modification layer should impart to the surface a low contact angle, typically ranging from

CA 02838176 2013-12-23 about 10 to 30 and usually from about 15 to 25° and a fast wicking time, e.g. 0.5 to 2 and usually from about 1 to 2 s, even after an extended period of time at an elevated temperature, e.g. even after 7 to 14 days at a temperature of from about 4 to 56° C. Generally, the selfassembling molecule that makes up the surface modification layer is a linear molecule having a sulfhydryl end group and a sulfonate end group separated by a lower alkyl linking group. The term sulfonate end group is used herein to refer to both a sulfonic acid moiety as well as a sulfonate moiety, which may be associated with a cation, e.g. sodium, as is found in a sulfonate salt. The alkyl linking group generally ranges from about 1 to 8, usually from about 1 to 6 carbon atoms in length, and may or may not include one or more sites of unsaturation, but is generally a saturated molecule. In certain embodiments, the number of carbon atoms in the alkyl linking group ranges from about 1 to 4 and often from about 1 to 3, with methylene and ethylene linking groups being common in these embodiments. In many embodiments, the molecule that makes up the self-assembling surface modification layer is a molecule of the formula:

HS-(CH₂)_n-SO₃Y wherein:

n is an integer from 1 to 6; and

Y is H or a cation.

Of particular interest are surface modification layers made up of 2-mercaptoethane ethane sulfonic acid or a salt thereof, e.g. 2-mercaptoethane sulfonate sodium. The working and reference electrodes may be fabricated using any convenient protocol. A representative protocol includes preparation of the metal electrodes by first sputtering the metal layer of sufficient thickness onto the surface of the inert backing material. Next, the electrode(s) to be surface modified, or at least the metallic surface that is to be modified, to have the surface modification layer is contacted with a fluid composition, e.g. an aqueous organic solution, of the self-assembling molecule. Contact may be achieved by any convenient means, including submersion slot coating, grevure printing of the electrode into the composition. The concentration of the self-assembling molecule in the fluid composition typically ranges from about 0.5 to 1%, usually from about 0.05 to 0.5% and more usually from about 0.05 to 0.3%. Contact is maintained for a sufficient period of time for the monolayer to form, e.g. for a period of time ranging from about 0.5 to 3 minutes, usually from about 0.5 to 2 min, followed by drying of the electrode surface for use in the electrochemical test strips. A

CA 02838176 2013-12-23 feature of the electrochemical test strips is that the working and reference electrodes as described above face each other and are separated by only a short distance. This minimal spacing of the working and reference electrodes in the test strips is a result of the presence of a thin spacer layer positioned or sandwiched between the working and reference electrodes. The thickness of this spacer layer generally ranges from about 1 to 500 um, usually from about 102 to 153 um. Further details of a suitable spacer layer can be found in the description of U.S. 6,475,372 provided above. The electrochemical test strips include a reaction zone or area that is defined by the working electrode, the reference electrode and the spacer layer, where these elements are described above. With respect to the reaction area and the redox reagent system present in the reaction area, reference can be had to the teachings of US 6,475,372, above, with respect to the specific enzyme and mediator component. Other reagents that may be present in the reaction area include buffering agents, e.g. citraconate, citrate, phosphate, Good buffers and the like. The redox reagent system is generally present in dry form. The amounts of the various components may vary, where the amount of enzyme component typically ranges from about 0.1 to 10% by weight.

In U.S. 6,620,310, a fluid sample is introduced into an electrochemical cell having oppositely spaced apart working and reference electrodes. An electric potential is applied to the cell to first achieve a steady state cell current. A decrease in the steady state cell cunent is then detected and related to a change in viscosity of the sample. The electrochemical test strips are made up of two opposing metal electrodes separated by a thin spacer layer, where these components define a reaction area or zone that makes up the electrochemical cell. The working and reference electrodes may be generally configured in the form of elongated rectangular strips. Typically, the length of the electrodes ranges from about 1.9 to 4.5 cm, usually from about 2.0 to 2.8 cm. The width of the electrodes ranges from about 0.07 to 0.76 cm, usually from about 0.24 to 0.60 cm. The working and reference electrodes typically have a thickness ranging from about 10 to 100 nm and usually from about 10 to 20 nm. At least the surface of the working and reference electrodes that faces the reaction area of the electrochemical cell in the strip is a metal, where metals of interest include palladium, gold, platinum, silver, iridium, carbon (conductive carbon ink), doped tin oxide, stainless steel and the like. In many embodiments, the metal is gold or palladium. While in principle the entire electrode may be made of the metal, each of the electrodes is generally made up of an inert support material on the surface of which is present a thin layer of the metal component of the

CA 02838176 2013-12-23 electrode. In these more common embodiments, the thickness of the inert backing material typically ranges from about 25 to 500, usually 50 to 400 pm, e.g., from about 127 to 178 pm, while the thickness of the metal layer typically ranges from about 10 to 100 nm and usually from about 10 to 40 nm, e.g. a sputtered metal layer. Any convenient inert backing material may be employed in the electrodes, as described above with respect to U.S. 6,475,372. The working and reference electrodes face each other and are separated by only a short distance, such that the distance between the working and reference electrodes in the reaction zone or area ofthe electrochemical test strip is extremely small. This minimal spacing of the working and reference electrodes in the test strips is a result of the presence of a thin spacer layer positioned or sandwiched between the working and reference electrodes. The thickness of this spacer layer may range from 50 to 750 pm and is often less than or equal to 500 pm, and usually ranges from about 100 to 175 pm, e.g., 102 to 153 pm. Further details of a suitable spacer layer can be found in the description of U.S. 6,475,372 provided above. The working and reference electrodes define the top and bottom of a reaction area, while the spacer layer defines the walls of the reaction area. The volume of the reaction area typically ranges from about 0.1 to 10 pL, usually from about 0.2 to 5.0 pL, and more usually from about 0.3 to 1.6 pL. The reaction area generally includes at least an inlet port, and in many embodiments also includes an outlet port, with dimensions suitably as described above with respect to U.S. 6,475,372. In many embodiments, a reagent system is present in the reaction area, where the reagent system interacts with components in the fluid sample during the assay. For example, in embodiments where the methods are used to detect a coagulation event, e.g., to measure PT of a sample, the reaction area or zone includes a reagent system that at least includes a redox couple, and often also includes a coagulation catalyzing agent. The redox couple of the reagent composition, when present, is made up of one or more redox couple agents. A variety of different redox couple agents are known in the art and include: ferricyanide, phenazine ethosulphate, phenazine methosulfate, pheylenediamine, 1-methoxyphenazine methosulfate, 2,6-dimethyl-l,4-benzoquinone, 2,5-dichloro-l,4-benzoquinone, ferrocene derivatives, osmium bipyridyl complexes, ruthenium complexes, and the like. In many embodiments, the reagent composition also includes a coagulation catalyzing agent. By coagulation catalyzing agent is meant one or more components or reactants that participate or interact with components present in the fluid sample, e.g., whole blood, to initiate the clotting process in the blood sample. For PT assays, the coagulation catalyzing agent generally comprises thromboplastin, which thromboplastin may be purified from a

CA 02838176 2013-12-23 naturally occurring source, e.g., an aqueous extract of acetone dried brain tissue, or synthetic recombinant thromboplastin (r-DNA thromboplastin), which generally includes purified recombinant tissue factor protein and a purified artificial lipid component. A representative coagulation catalyzing agent is thromboplastin-XS with calcium sold under the trade name INNOVIN® by Dade International, Miami Fla. Other reagents that may be present in the reaction area include buffering agents, e.g. citraconate, citrate, malic, maleic, phosphate, Good buffers and the like. Yet other agents that may be present include: divalent cations such as calcium chloride, and magnesium chloride; surfactants such as Triton, Macol, Tetronic, Silwet, Zonyl, and Pluronic; stabilizing agents such as albumin, sucrose, trehalose, mannitol, and lactose. The reagent system, when present, is generally present in dry form. The amounts of the various components may vary, where the amount of the oxidized redox couple component typically ranges from about 5 to 1000 mM, usually from about 90 to 900 mM; the reduced redox couple component typically ranges from about 1 to 20 mM, usually from about 5 to 15 mM; the amount of buffer typically ranges from about 0 to 300 mM, usually from about 50 to 100 mM; and the amount of coagulation catalyzing agent component typically ranges from about 0.005 to 50 mg/cm², usually from about 0.05 to 5 mg/cm². The overall mass of dry reagent present in the reaction area or zone in these embodiments generally ranges from about 4 to 700 ng/cm², usually from about 8 to 350 ng/cm².

In U.S. 6,558,528, electrochemical test strip cards that can be singulated to produce electrochemical test strips are taught. The electrochemical test cards are made up of two or more electrochemical test strip precursors, where each precursor is characterized by the presence of a dry reagent housing reaction chamber bounded by opposing electrodes. In gaseous communication with each reaction chamber of the card is an integrated desiccant. The electrochemical test strip cards can be singulated or cut into individual electrochemical test strips. The electrochemical test strips include two opposing metal electrodes separated by a thin spacer layer, where these components define a reaction chamber, i.e., area or zone, in which is located a redox reagent system. The working and reference electrodes are generally configured in the form of elongated rectangular strips. Typically, the length of the electrodes ranges from about 1.9 to 4.5 cm, usually from about 2.0 to 2.8 cm. The width of the electrodes ranges from about 0.38 to 0.76 cm, usually from about 0.51 to 0.67 cm. The reference electrodes typically have a thickness ranging from about 10 to 100 nm and usually

CA 02838176 2013-12-23 from about 10 to 20 nm. At least the surface of the working and reference electrodes that faces the reaction area in the strip is a metal, where metals of interest include palladium, gold, platinum, silver, iridium, carbon, doped tin oxide, stainless steel and the like. While in principle the entire electrode may be made of the metal, each of the electrodes is generally made up of an inert support material on the surface of which is present a thin layer of the metal component of the electrode, as described above with respect to US 6,475,372. A feature of the electrochemical test strips produced from the cards is that the working and reference electrodes face each other and are separated by only a short distance. This minimal spacing of the working and reference electrodes in the test strips is a result of the presence of a thin spacer layer positioned or sandwiched between the working and reference electrodes. The thickness of this spacer layer generally should be less than or equal to 500 pm, and usually ranges from about 102 to 153 pm. Further details of a suitable spacer layer can be found in the description of U.S. 6,475,372 provided above. The electrochemical test strips include a reaction chamber, zone or area that is defined by the working electrode, the reference electrode and the spacer layer. With respect to the reaction area and the redox reagent system present in the reaction area, reference can be had to the teachings of US 6,475,372, above, with respect to the specific enzyme, mediator component and other reagents. The redox reagent system is generally present in dry form. The electrochemical test strip cards may be fabricated using any convenient procedure. Various layers of different materials, e.g., electrode layers, spacer layers, etc., may be brought together into a single card format, which is then laminated in a barrier material to produce the final product. The cards may be fabricated using any convenient protocol. In a suitable process, the initial starting materials are top electrode layer, bottom electrode layer and middle spacer layer. In one example, top electrode layer is a PET substrate with a sputtered gold layer on the bottom, while the bottom electrode layer is a PET substrate with a sputtered palladium layer on the top. Reagents are coated onto the bottom layer. Spacer layer is a 3-layer lamination of PSA/PET/PSA (PSA=pressure sensitive adhesive; PET=polyester terepthalate) which has the precursor fluid pathways and reaction chamber present. These three layers are laminated together to produce structure and a hole is punched through the composite laminate structure to produce a desiccant chamber. Punching of the desiccant chamber also results in the production of a fluid stop junction downstream from the reaction chamber which serves to precisely limit the amount of fluid sample that enters the strip upon use. A desiccant material, e.g., block, beads, etc., is then positioned in the punched out desiccant chamber and

CA 02838176 2013-12-23 the resulting structure is laminated or sealed between top and bottom barrier layers and consisting of, e.g., PSA-faced aluminum film to produce the final card. If the layers and are sufficiently malleable, the film will deform during lamination to allow for the thickness of the desiccant. If the materials can be embossed, they may be embossed prior to lamination to form a pocket which accepts the desiccant material. At the end of card is an information storage means, e.g., barcode, transmitter, etc., which provides information such as calibration information to the meter with which the card is employed. The configuration of the various electrode layers provides for electrical contacts in the final strips singulated from the cards.

. Such test strips are used with meters configured for electrochemical measurements, such as those disclosed in the above-identified patent references. Electrochemical biosensors other than test strips may also be suitable for use with the present invention. For example, the electrochemical cell may have a cylindrical configuration wherein a core electrode is coaxially positioned within a second tubular electrode. Such electrochemical cell configurations may be in the form of micro-needles and, as such, are either integral within the needle structure for in situ (e.g., typically under the skin surface) measurements or otherwise in physical or fluid communication with a micro-needle structure. Examples of such micro-needle are disclosed in copending U.S. Patent Nos 6,793,632 and 6,501,976. In 6,793,632, the device has at least one micro-needle having an open distal end used to penetrate the skin to a depth where pain and bleeding are minimized. The device further includes a hydrophilic gel within the micro-needle for sampling the biological fluid constituents and an electrochemical cell for measuring the concentration of targeted constituents within the sampled biological fluid constituents. In 6,501,976, the subject devices include a sampling device configured to pierce a skin surface to provide access to biological fluid and concentrically-spaced working and reference electrodes positioned within the elongated sampling device that define an electrochemical cell for measuring the concentration of analyte within the biological fluid. For purposes of this disclosure, the subject devices will be described in use with electrochemical cells in test strip configurations; however, those skilled in the art will appreciate that the subject devices may be used with any suitable electrochemical cell configuration, including micro-needle configurations.

The type of electrochemical measurement that is made may vary depending on the

CA 02838176 2013-12-23 particular nature of the assay and the meter with which the electrochemical test strip is employed, e.g., depending on whether the assay is coulometric, amperometric or potentiometric. The electrochemical cell will measure charge in a coulometric assay, current in an amperometric assay and potential in a potentiometric assay. For purposes of this disclosure, the present invention will be described in the context of amperometric assays; however, the subject devices may be employed with any type of assay and electrochemical measurement.

Generally, in any configuration, an electrochemical cell includes at least two electrodes spaced-apart in either a facing arrangement or in a side-by-side arrangement in the 10 same plane. In the first arrangement, the electrodes are separated by a thin spacer layer, which defines a reaction area or zone, or chamber into which a biological sample is deposited or transferred for analyte concentration measurement. In the side-by-side configuration, the electrodes are in a chamber with a defined thickness and volume. Present in the reaction area or chamber, i.e., coated on one or more of the facing surfaces of the electrodes, are one or more redox reagents selected to chemically react the target analyte(s). Such redox reagents typically comprise at least one enzyme and a mediator.

A representation of an exemplary conventional electrochemical test strip 2 suitable for use with the present invention is provided in the exploded view of Fig. 1. Test strip 2 is made up of a two electrodes 4, 8 separated by a spacer layer 12 which has a cutaway section 20 that defines the reaction zone or area 14. Generally, the electrodes 4, 8 are configured in the form of elongated rectangular strips each having a length in the range from about 2 to 6 cm, usually from about 3 to 4 cm, having a width in the range from about 0.3 to 1.0 cm, usually from about 0.5 to 0.7 cm, and having a thickness in the range from about 0.2 to 1.2 mm, and usually from 0.38 to 0.64 mm.

The surfaces of electrodes 4, 8 that face the reaction area in the strip is made of a conductive material, preferably a metal, where metals of interest include palladium, gold, platinum, iridium, doped indium tin oxide, stainless steel, carbon and the like. The outside surfaces 6,10 of electrodes 4, 8 are made of an inert support or backing material. Any suitable inert backing material may be used with electrodes 4, 8, where typically the material is a rigid material that is capable of providing structural support to the electrode and, in turn, the electrochemical test strip as a whole. Such suitable materials include plastics, e.g., PET,

CA 02838176 2013-12-23

PETG, polyimide, polycarbonate, polystyrene, silicon, ceramic, glass, and the like. Electrodes 4, 8 and test strip 2 may be fabricated using any of various manufacturing techniques known to those skilled in the relevant art.

As described above, a thin spacer layer 12 is positioned or sandwiched between electrodes 4, 8. The thickness of spacer layer 12 generally ranges from about 1 to 500 pm, and usually from about 50 to 150 pm. Spacer layer 12 may be fabricated from any convenient material, where representative suitable materials include PET, PETG, polyimide, polycarbonate and the like. The surfaces of spacer layer 12 may be treated so as to be adhesive with respective electrodes 4, 8 and thereby maintain the structure of the electrochemical test strip 2.

Spacer layer 12 is cut so as to provide a reaction zone or area 14 having any appropriate shape including circular, square, triangular, rectangular, or irregular shaped reaction areas. The top and bottom of the reaction zone 14 is defined by the facing surfaces of electrodes 4, 8 while spacer layer 12 defines the side walls of the reaction area 14. The volume of the reaction area ranges from at least about 0.1 to 10 pl, usually from about 0.2 to 5.0 pL and more usually from about 0.3 to 1.6 pL.

Present in the reaction area 14 is a redox reagent system, which reagent system provides for the species that is detected by the electrode and therefore is used to derive the concentration of analyte in a biological sample. The redox reagent system present in the reaction area typically includes at least an enzyme(s) and a mediator. In many embodiments, the enzyme member(s) of the redox reagent system is an enzyme or plurality of enzymes that work in concert to oxidize the analyte of interest. In other words, the enzyme component of the redox reagent system is made up of a single analyte oxidizing enzyme or a collection of two or more enzymes that work in concert to oxidize the analyte of interest. Typical enzymes of interest include oxidoreductases, hydrolases, transferases and the like; however, the specific enzyme present in the reaction area depends on the particular analyte for which the electrochemical test strip is designed to detect. Where the analyte of interest is glucose, for example, suitable enzymes include glucose oxidase, glucose dehydrogenase (either βnicotineamide adennine dinucleotide based (NAD) or 4,5-Dihydro-4,5-dioxo-lHpyrrolo[2,3-f]quinoline-2,7,9-tricarboxyIic acid based (PQQ)). Where the analyte is cholesterol, suitable enzymes include cholesterol esterase and cholesterol oxidase. For other

CA 02838176 2013-12-23 analytes, enzymes including but not limited to lipoprotein lipase, glycerol kinase, glycerol-3phosphate oxidase, lactate oxidase, lactate dehydrogenase, pyruvate oxidase, alcohol oxidase, bilirubin oxidase, uricase, and the like may be used.

The second component of the redox reagent system is a mediator component, which is made up of one or more mediator agents. A variety of different mediator agents are known in the art and include: ferricyanide, phenazine ethosulphate, phenazine methosulfate, pheylenediamine, 1-methoxy-phenazine methosulfate, 2,6-dimethyl-l, 4-benzoquinone, 2,5dichloro-l,4-benzoquinone, fenocene derivatives, osmium bipyridyl complexes, ruthenium complexes and the like. In those embodiments where glucose is the analyte of interest and glucose oxidase or glucose dehydrogenase is the enzyme components, mediator of particular interest is ferricyanide. Other reagents that may be present in the reaction area include buffering agents, e.g., citraconate, citrate, phosphate, “Good” buffers and the like.

The redox reagent system is generally present in dry form. The amounts of the various components may vary, where the amount of enzyme component typically ranges from about 0.1 to 20% by weight.

Methods of the Present Invention

As summarized above, the subject invention includes methods for determining the volume of biological sample deposited or transferred to an electrochemical cell having a reaction zone or chamber with a defined volume and determining whether such volume is adequate to produce an accurate analyte concentration measurement. As mentioned above, a feature of the subject methods in determining sample volume is the determination of the equivalent capacitance of the cell, as well as the equivalent cell resistance. As such, the subject methods provide a more accurate measure of sample volume than that which has been achieved by the prior art.

Another feature of the subject methods in determining the equivalent cell capacitance and resistance and sample volume is to disregard certain characteristics or factors of the sampled solution or ambient conditions which either have no affect on the determination of the equivalent capacitance and/or equivalent resistance or are otherwise strictly controlled so as not to have such an affect. Such factors which are controlled or independent of the equivalent capacitance include but are not limited to the concentration of 22

CA 02838176 2013-12-23 ionic species, blood hematocrit, blood glucose concentration, environmental temperature, the blood donor, and sensor interferences typically found in blood, cell thickness and biosensor aging.

For purposes of understanding the description of the subject methods, a simplified model of an impedance circuit 40 of the electrochemical cell of the test strip of Fig. 1 is provided in Fig. 2. Impedance circuit 40 is representative of the impedance factors of the test strip when containing a sample of biological solution and having a voltage applied to it. When both AC and DC voltages are applied to the cell, impedance circuit 40 comprises equivalent cell capacitance (C) 42, which includes the double layer and the Faradaic capacitances, and the equivalent cell resistance (R) 46 of the electrochemical cell.

Prior to practicing the subject methods, it is first necessary to obtain the biological sample to be measured and placing such sample within the test strip cell. Placement of the sample within the test strip may be accomplished by first inserting the test strip into the test meter and then applying the sample to the test strip (“on-meter dosing”), or by first applying the sample to the test strip and then inserting the test strip into the test meter (“off-meter dosing”). The latter sequence is often preferred in hospital environments as it is more likely to cross-contamination within the meter. The measurement meter then detects that the biological sample has been introduced into the electrochemical cell (as disclosed in U.S. Patent No. 6,193,873).

Once such sample is detected, the first step of the subject methods is to apply an alternating voltage (V_Ac) of low amplitude to the biosensor. The amplitude of the applied AC voltage is selected such that its application does not result in a Faradaic current (i_F), i.e., the electron transfer that occurs at the electrode surface as result of electrochemical reaction upon application of a voltage to the electrode. As such, the amplitude of the applied AC voltage is in the range from about 2 to 100 mV rms, typically is in the range from about 5 to 75 mV rms, and more typically is about 50 mV rms. The frequency (f) of the applied AC voltage is also selected so as to maximize the signal to noise ratio (i.e., the ratio of the measured equivalent cell capacitance to the variability in the measured equivalent cell capacitance) of the cell. As such, an acceptable frequency of the applied AC voltage is in the range from about 50 to 10,000 Hz, typically in the range from about 75 to 1,000 Hz, and more typically is about 100 Hz.

CA 02838176 2013-12-23

Optionally, a DC voltage may be applied to the biosensor, either simultaneously with the AC voltage or fractions of a second prior to application of the AC voltage This DC voltage is generally in the range from about 0 to 600 mV, typically from about 200 to 500 mV and more typically from 300 to 400 mV. The DC voltage component causes the electrochemical cell equivalent capacitance to stabilize more rapidly than it would with an AC voltage alone. The rapid stabilization of the cell capacitance allows a determination of the sample volume before commencing the analyte measurement, thus minimizing overall testing time.

After application of the AC and DC voltages, the alternating current (i_Ac) generated from charging the electrochemical cell is then measured. The equivalent cell capacitance of the biosensor can then be determined from the amplitude and the phase of the resulting alternating current.

For the equivalent cell capacitance to depend only on the cell surface area contacted by biological sample, the charge separation distance (ddi) of the double layer capacitance 15 must be strictly controlled such that it is constant during the determination of the volume of the sample solution. The thickness of the double layer capacitor is dependent on the applied voltage, concentrations of the charged species in the solution. In order to ensure a constant thickness of the charge separation layer, and thus ensure an accurate determination of the surface area of each electrode in contact with the sample solution and an accurate determination of the sample volume, the ion concentration of the redox reagent is strictly controlled, while the ion concentration of the sample is physiologically controlled in a very narrow range.

As mentioned above, the equivalent resistance of the electrochemical cell is additionally relevant to accurately determining sample volume. Thus, establishing that the 25 equivalent cell resistance (R) is inversely proportional to the surface area of the cell covered by the sample and that the equivalent cell capacitance (C) is linearly proportional to the covered cell area, as represented by the following relationships:

R = pl/A and C oc A where p is the resistivity of the electrochemical cell, 1 is the length of the cell electrode, and A is the conducting surface area of the cell, the following relationship exists:

CA 02838176 2013-12-23

C/Roc A²

Thus, the amount of surface area of the cell covered by the biological sample can be determined by the ratio of the equivalent cell capacitance to the equivalent cell resistance. By taking into consideration the effect of sample volume on two impedance components (capacitance and resistance) of the cell rather than just on one of them (e.g., capacitance), a relationship is established which offers more sensitivity in response to the variations in the amount of cell surface area covered by a biological sample. More specifically, the ratio of the cell capacitance to cell resistance provides a quadratic relationship rather than a linear relationship with respect to the surface area of the cell covered by a biological sample, thus increasing the sensitivity in the measured change(s) to the covered cell area. Therefore, the determination of the covered cell area according to this method is even more accurate than when only the equivalent cell capacitance is considered.

Upon a determination of the surface area of the electrode in contact with or covered by the sample solution, the volume (V_s) of the sample solution within the biosensor, i.e., within the reaction zone of the electrochemical cell, can then be determined according to the following equation:

V_s = A«d where d is the distance between the cell electrodes in a facing electrode configuration or the depth of the cell in a side-to-side electrode configuration.

A determination is then made as to whether the volume of the sample provided to the test strip is adequate to proceed with the analyte concentration measurement. Such a determination is made by comparing the calculated sample volume with the total volume of the electrochemical cell. As is discussed in more detail below with respect to the systems of the present invention, certain parameters including but not limited to the value of the total cell volume, operating temperature range, proper test strip insertion into the meter, among other data (both static and dynamic) or parameters related to the particular cell, are stored in the memory of a microprocessor, for example, upon calibration of the meter and other related components of the subject systems.

If the sample volume is determined to be adequate, measurement of the desired

CA 02838176 2013-12-23 characteristic, e.g., an analyte concentration, is made, the results of which may be displayed on a display unit, described in more detail below with respect to the subject systems. On the other hand, if the sample volume is determined to be inadequate, i.e., too low, to provide an accurate measurement, the display unit may be configured to display a low volume icon.

As discussed above, certain embodiments of the subject methods include the additional function of compensating for an inadequate sample solution volume in order to make an accurate measurement of the selected characteristic, e.g., concentration of the targeted analyte(s), without having to redo the sampling and testing steps.

It is known in the art that the concentration of a selected analyte, such as glucose, of the biological sample within the cell is proportional to the Faradaic current (Ip) that is passed through the electrochemical cell when a DC voltage is applied, that the cell current is proportional to the cell surface area covered by the sample solution. As mentioned above, the inventors have determined that such surface area is proportional to the equivalent capacitance of the cell. Thus, the concentration of the selected analyte is proportional to the equivalent cell capacitance. By determining the equivalent cell capacitance when a sample solution is present and by knowing the capacitance of the cell when completely filled with a biological solution (determined by a calibration process), the compensation factor (F_cf) necessary to compensate for a low sample volume and to provide an accurate analyte concentration measurement can be determined according to the following equation:

F_cf=C_f/C_pf where Cf is the equivalent capacitance of the completely filled electrochemical cell and C_Pf is the equivalent capacitance of the electrochemical cell containing the inadequate volume of biological sample. The corrected analyte concentration measurement (G) is then made with the appropriate compensation factor (F_cf) according to the following equation:

G = F_Cf · G_Pf where G_Pf is the analyte concentration calculated from the cell containing inadequate volume of biological sample. In being able to compensate for inadequately low sample volume, the subject methods avoid wasting test strips, decrease costs and reduce the time necessary for conducting the analyte measurement.

CA 02838176 2013-12-23

Thus, generally summarized in accordance with the above principles and discoveries, certain methods of the present invention include the steps of applying an AC voltage having a selected amplitude and a selected frequency to the biosensor with or without also applying a DC voltage to the biosensor; measuring the AC current generated by application of the voltage(s); determining the capacitance or both the capacitance and the resistance ofthe biosensor from the measured AC current; determining the surface area of the portion of the biosensor in contact with the sample based on the determined capacitance or based on both the determined capacitance and the determined resistance; and then determining the volume of the sample within the biosensor based on the determined surface area.

Other subject methods further include the step of measuring one or more physical or chemical characteristics of the biological sample, such as the concentration of one or more selected analytes, based on a determination that the sample volume is adequate. Still other subject methods include compensating for an inadequate volume of a biological sample held within an electrochemical biosensor for measurement of at least one characteristic of the biological sample in order to accurately measure the value of the characteristic. Such compensation method includes determining the necessary compensation factor to compensate for an inadequate sample volume if such is determined, and thereafter compensating for the inadequate sample volume while measuring, for example, the concentration of a selected analyte present within the sample. The step of determining the necessary compensation factor includes determining the ratio of the equivalent capacitance of the biosensor when completely filled with the sample to the determined equivalent capacitance of the biosensor with the inadequate sample volume. The value of the equivalent capacitance of the biosensor when completely filled within said sample may be accessed from a memory storage means.

Systems of the Present Invention

The present invention also provides systems for practicing the above-described subject methods. Generally described, such a system may include the following components for carrying out the steps of the above-described methods of the present invention: a voltage supply configured for applying a voltage to the electrochemical cell, means for measuring a

CA 02838176 2013-12-23 current generated by the cell when the voltage is applied to the cell, means for deriving the capacitance and/or resistance of the cell from the measured current; means for deriving the surface area of the cell covered by the biological sample from the cell capacitance and/or resistance; and means for deriving the volume of the biological sample from the cell surface area. Certain systems further include means for determining whether the sample volume is adequate for making an accurate measurement of one or more selected characteristics of the biological sample, including but not limited to the concentration of one or more selected analytes within the biological sample. Certain of these systems further include means for compensating for an inadequate sample volume while the selected characteristic of the biological sample.

Fig. 3 illustrates a block diagram of an exemplary system 50 of the present invention. System 50 includes electronic components and circuitry configured to be electronically coupled to a biosensor 70, e.g., an electrochemical measurement cell in the form of a disposable test strip as described above with respect to Fig. 1, into which the sampled biological solution to be tested is deposited or transferred. More particularly, system 50 includes a voltage supply 52 for supplying the requisite AC and DC input voltages to biosensor 70. System 50 further includes a current- to-voltage converter 54, an analog to digital converter 56 and a microprocessor 58, which collectively act to receive and process data from biosensor 70. In particular, current-to-voltage converter 54 is operatively coupled to an output terminal of biosensor 70 for receiving the output current signal from biosensor 70 when a voltage is applied by voltage supply 52 and for converting that current signal to a corresponding voltage signal. This corresponding voltage signal is then provided as an input to analog-to-digital converter 56 which converts the analog voltage signal to a corresponding digital value. This digital voltage value is then provided as an input signal to microprocessor

54 which is programmed to derive and/or determine the factors or parameters of interest,

e.g., equivalent cell capacitance, equivalent cell resistance, the surface area of the biosensor in contact with the biosensor, the volume of the biological sample, the volume compensation factor, etc.; and to control the timing of each of these functions. As previously mentioned, microprocessor 54 may include a memory storage means for storing predetermined, preselected or calibrated data or information such as the total volume of the electrochemical cell, calibration parameters, operating temperature range, sample type information, sample detection information and the like which are necessary or useful for performing the steps and

CA 02838176 2013-12-23 functions of the subject methods. Although a microprocessor has been described for purposes of storing and processing data in accordance with the principles of the present invention, those skilled in the art will recognize that other discrete electronic components may be collectively configured to achieve the objectives of the present invention.

The subject system may further include a display device or unit 60 for displaying selected empirical or symbolic data, information or outputs supplied by the control device or microprocessor. Such data, information or outputs may include, but are not limited to, measured or derived values of selected output signals and impedance factors, sample volume size, volume adequacy/inadequacy indicator icons, inadequate volume compensation factors, concentrations of analytes of interest, biological sample versus control sample indicator icons, calibration results, etc.

In many embodiments, the electrochemical signal application, measurement, derivation, calculation, compensation and display steps, as described above, are performed automatically by the subject systems designed to work with the electrochemical cell. As such, the electronic circuitry of the subject systems may be structurally and functionally integrated into a meter or other automated device configured to receive and operatively engage with an electrochemical cell, e.g., a disposable test strip, and to measure one or more physical or chemical characteristics of a biological sample held within the electrochemical cell. Most typically, such characteristics include the concentration of one or more target analytes within the biological sample. A representative meter or device for automatically practicing some of the same or similar steps and functions, such that the user need only apply a biological sample to the reaction zone of an electrochemical cell and then read the final analyte concentration result from the device, is further described in U.S. Patent No. 6,193,873 Bl. For example, in an embodiment of suitable circuitry, a constant current source is applied to the strips with switch in a position 1. The current source consists of operational amplifier, voltage reference, and resistors. The current is determined by the ratio of voltage reference to resistor. Resistor is used to generate the required bias. Operational amplifier and resistor are used as a current-to-voltage converter. Initially, with no sample on the strip, the resistance between points is very large, and the current passing through the strip is negligible. The output voltage of operational amplifier (VI) is high in this condition. When a sample is applied to the strip, its resistance drops significantly and, since a constant current flows through the strip, VI drops. VI is fed to a microprocessor through analog-to-digital

CA 02838176 2013-12-23 converter. The microprocessor, recognizing this reduced voltage as sample detection, switches to a position 2 to disconnect the strip from the current source and connect it to the voltage source. In this condition, a chronoamperometric measurement can be achieved by measuring the output voltage of the operational amplifier (V2). This voltage is proportional to the current passing through the strip.

Those skilled in the relevant art will appreciate that the subject systems are usable with assay systems that do not comprise biosensors of the type described above. Such other systems include, for example, an electrochemical cell having at least two electrodes and a redox reagent system having a fixed concentration of ions, wherein the electrodes are configured to be placed within a biological sample or environment having a fixed concentration of ions.

Examples

The following results have been observed in connection with the present invention. Figs. 4-8 illustrate the variation in the experimental results of measurements made of test strips having an adequate sample volume (i.e., test strips having reaction zones completely filled with sample solution or material) and of test strips having less than an adequate sample volume (i.e., test strips having reaction zones half filled with sample solution or material). More particularly, measurements of the equivalent capacitance of the electrochemical cells and the sample solution resistance were made over selected periods of time during which the appropriate voltages were applied to the test strips. The following experimental results are offered by way of illustration and not by way of limitation. The results illustrated were collected by dosing the strips with 5 different blood donors, glucose range of 40-600 mg/dL, hematocrit of 20% and 70%, at room temperature.

Fig. 4 illustrates a comparison of the change in the equivalent cell capacitance (yaxis) over time (x-axis) of test strips 130 completely filled with blood samples and test strips 132 half-filled with blood samples. The graph shows that the completely filled test strips 130 produced an equivalent cell capacitance twice as great as the equivalent capacitance of the half-filled test strips 132.

Fig. 5 illustrates a comparison in the change in the equivalent cell resistance (y-axis) over time (x-axis) in test strips 136 completely filled with the blood samples and test strips

CA 02838176 2013-12-23

134 half-filled with the blood samples. The graph shows that the greater volume of blood sample produced an equivalent cell resistance about one half of that of the test strips filled with the smaller volume of blood sample.

Fig. 6 illustrates histograms of the equivalent cell capacitance (x-axis) of test strips 140 completely filled with blood samples and test strips 138 half-filled with blood samples, wherein the equivalent cell capacitance is measured at 0.5 seconds after blood sample is applied to the test strip. The graph shows that the completely filled test strips 140 produced an equivalent cell capacitance twice as great as the equivalent capacitance of the half-filled test strips 138 with a very good reproducibility.

Fig. 7 illustrates a comparison of the change in the ratio of the equivalent cell capacitance to the equivalent cell resistance (C/R) (y-axis) over time (x-axis) of test strips 142 completely filled with blood samples and test strips 144 half-filled with blood samples. The graph shows that the completely filled test strips 142 produced a C/R about four times as great as the C/R of the half-filled test strips 144.

Fig. 8 illustrates a comparison of a scatter diagram of the ratio of the equivalent cell capacitance to the equivalent cell resistance (C/R) (y-axis) versus the equivalent cell capacitance (x-axis) of test strips 146 completely filled with blood samples and test strips 148 half-filled with blood samples, wherein the capacitance and the resistance are measured at 0.5 seconds after application the blood sample to the test strip. The graph shows that C/R produced more sensitivity to the sample volume compared to the equivalent cell capacitance C.

Kits

Also provided by the subject invention are kits for use in practicing the subject methods. The kits of the subject invention include a subject system including the electronic circuitry, as described above, or in the form of a meter or other automated instrument, as described above, for determining whether the volume of sample applied to a test strip is adequate enough to provide an accurate analyte concentration measurement to be made. In certain other kits, the subject systems also compensate for such inadequate volume when making an analyte concentration measurement. The kits may further include instructions for using the subject systems according to the subject methods with an electrochemical cell,

CA 02838176 2015-09-03 in the form of a test strip or micro-needle or the like, in the determination of the volume of a sampled solution or material held within the electrochemical cell. These instructions may be present on one or more of the packaging, a label insert, and the like.

It is evident from the above description that the features of the subject methods and systems overcome many of the disadvantages of prior art techniques for determining the volume of a biological sample deposited on a test strip for electrochemical analyte concentration analysis, and provide certain advantages including, but not limited to, providing a very accurate means and technique for making such sample volume determination and decreasing the time necessary to conduct analyte concentration measurements. Further, such sample volume determination is not subject to variations of blood glucose concentration, blood hematocrit level, the blood donor, testing temperature, and the concentration of interferences often present in blood samples. Other advantages of the invention include the ability to compensate for an inadequate sample volume and proceed with the analyte concentration measurement without having to abort the testing procedure, minimizing waste and costs. As such, the subject invention represents a significant contribution to the field of fluid of biological sample volume determination and analyte concentration measurement.

The subject invention is shown and described herein in what is considered to be the most practical and preferred embodiments. It is recognized, however, that departures may be made there from, which are within the scope of the invention, and that obvious modifications will occur to one skilled in the art upon reading this disclosure. The scope of the claims may be given the broadest interpretation consistent with the description as a whole.