HK1131872B - Transient decay amperometry - Google Patents

Description

Transient decay amperometry

Reference to related applications

Priority of U.S. provisional application No.60/854,060 entitled "Transient Decay Amperometry" filed 24.10.2006, the entire contents of which are incorporated herein by reference, priority of U.S. provisional application No.60/869,557 entitled "Transient Decay Amperometry" filed 11.12.2006, the entire contents of which are incorporated herein by reference, and priority of U.S. provisional application No.60/869,625 entitled "Transient Decay Amperometry" filed 12.2006, the entire contents of which are incorporated herein by reference.

Background

Biosensors provide for the analysis of biological fluids such as whole blood, urine, or saliva. In general, biosensors analyze a sample of a biological fluid to determine the concentration of one or more analytes, such as alcohol, glucose, uric acid, lactate, cholesterol, or bilirubin, in the biological fluid. Such analysis can be used to diagnose and treat physiological abnormalities. For example, a diabetic patient may use a biosensor to measure glucose levels in whole blood to adjust diet and/or medication.

Biosensors may be implemented with desktop devices, portable devices, and similar measurement devices. The portable measuring device may be hand-held. Biosensors may be designed to analyze one or more analytes, and may use different amounts of biological fluids. Some biosensors may analyze a drop of whole blood, for example, a volume of 0.25-15 microliters (μ L) of whole blood. Examples of portable measuring devices include: ascensia available from Bayer CorporationAnda measuring instrument; available from Abbott, Abbott Park, IllinoisA biosensor; available from Roche of Indianapolis, IndianaA biosensor; and OneTouch available from Lifescan of Milpitas, CaliforniaA biosensor. Examples of bench-top measuring devices include: BAS 100B Analyzer available from BAS Instruments of West Lafayette, Indiana; electrochemical workbench available from CH Instruments of Austin, Texas; another electrochemical workstation available from Cypress Systems, Lawrence, Kansas; and EG available from Princeton Research Instruments of Princeton City, N.J.&G electrochemical instrument.

Biosensors typically measure an electrical signal to determine the analyte concentration in a sample of a biological fluid. When an input signal is applied to the sample, the analyte typically undergoes an oxidation/reduction reaction or a redox reaction. An enzyme or similar species may be added to the sample to enhance the redox reaction. The input signal is typically an electrical signal, such as a current or a potential. The redox reaction generates an output signal in response to an input signal. The output signal is typically an electrical signal, such as a current or potential, which can be measured and correlated to the analyte concentration in the biological fluid.

Many biosensors include a measuring device and a sensor strip. The sensor strip may be used in vitro, in vivo, or in part in vivo in a living organism. When used outside of the living organism, a sample of biological fluid is introduced into a sample reservoir in the sensor strip. The sensor strip may be placed in the measurement device for analysis before, after, or during introduction of the sample. When in vivo or in part of a living organism, the sensor strip may be continuously immersed in the sample, or the sample may be intermittently introduced into the sensor strip. The sensor strip may include a reservoir that partially isolates a volume of sample or is open to the sample. Similarly, the sample may flow continuously through the sensor strip or be interrupted for analysis.

The measuring device typically has electrical contacts that connect with electrical conductors in the sensor strip. The electrical conductors are typically connected to working, counter, and/or other electrodes that extend into the sample reservoir. The measurement device applies an input signal to the electrical conductors in the sensor strip via the electrical contacts. Electrical conductors convey the input signal via the electrodes into the sample deposited in the sample reservoir. The redox reaction of the analyte generates an output signal in response to the input signal. The measurement device determines an analyte concentration in response to the output signal.

The sensor strip may include a reagent that reacts with an analyte in the biological fluid sample. The reagents may include an ionizing agent for promoting a redox reaction of the analyte, and a mediator or other substance that facilitates electron transfer between the analyte and the conductor. The ionizing agent may be an oxidoreductase, such as an analyte-specific enzyme, which catalyzes the oxidation of glucose in a whole blood sample. These reagents may include a binder for holding the enzyme and mediator together.

Many biosensors use amperometric methods in which an electrical signal having a constant potential (voltage) is applied to an electrical conductor of a sensor strip, and the measured output signal is a current. Thus, in an amperometric system, the current may be measured while applying constant potentials to the working and counter electrodes of the sensor strip. The measured current can then be used to determine the presence of and/or quantify the analyte in the sample. Amperometry measures the rate at which a measurable species, and therefore an analyte, is oxidized or reduced at the working electrode. In addition to the analyte, for example, the biological matrix and the mediator may also serve as a measurable species.

As the time for applying the input signal to the sensor strip increases, the rate at which the measurable species is oxidized or reduced at the working electrode decreases. Thus, after an initial period with a high current output, the recorded current from the sensor strip decreases as the input signal is continually applied. This decrease in current over time may be referred to as electrochemical decay, and the rate of this decay may be related to the concentration of the measurable species and thus the analyte in the sample. The electrochemical decay may be a transient or Cottrell (Cottrell) decay.

For example, electrochemical decay may be correlated to the analyte concentration in a sample by expressing the decay in an equation describing a line that relates current to time by a natural log function (ln). Thus, the output current may be expressed as a function of time with an exponential coefficient, where a negative exponential coefficient indicates the decay process. After the initial decrease in current output, the rate of decrease may remain relatively constant or continue to fluctuate.

U.S. Pat. No. 5,942,102 ("the' 102 patent") describes the relationship between measured output current and time during conventional analysis. An electrical signal is input to the sensor strip at about 60 seconds after the whole blood sample is introduced to the strip. Initially, a rapidly decreasing current is observed, followed by a relatively constant or "steady state" current output resulting from feedback of mediator from the counter electrode to the working electrode. The feedback of the mediator provided by the shorter distance between the electrodes results in the current becoming substantially unaffected by time after the initial decrease. In such conventional analysis, the current can be measured as a function of time by (1); and then (2) estimating the steady state current to determine the mediator concentration and the diffusion coefficient to determine the analyte concentration of the sample.

Although the analytical method described in the ' 102 patent relies on the steady-state portion of the current decay, U.S. Pat. Nos. 6,153,069 ("' 069 patent") and 6,413,411 ("' 411 patent") describe methods for determining the concentration of a mediator, and thus a base analyte, based on the diffusion coefficient of the mediator. These systems are configured to provide a rate of current decay described by the Cottrell equation.

The current measurement shows the Cottrell decay when the measured current is inversely proportional to the square root of time. The current measurement with Cottrell decay can be described by the following Cottrell equation given as equation (1):

wherein i is the measured current; c^bIs in mol/cm³Volume concentration of electrochemically active material in units; a is in cm²Electrode area in units; f is the Faraday constant of 96,500 Coulomb/equivalent; n is the number of electrons transferred in equivalents/mole; d is in cm²Diffusion coefficient in units of/sec; t is the electrochemical reaction time in seconds. Thus, the Cottrell equation describes the current as an exponential function of time, with a decay constant or exponential coefficient of-0.5. Electrochemical Methods available in Bard and Faulkner: fundFurther details regarding the required boundary conditions for the Cottrell equation and the Cottrell behavior are found in chapter 5, pages 136 to 145 of ametalsand Applications (1980).

Systems designed to operate according to the Cottrell current decay require a decay constant of-0.5. An electrochemical system exhibiting a decay constant of-0.5 suggests that there is a requirement for a Cottrell current, i.e., that the analyte has been completely converted to a measurable species and that the measurable species is a substantially constant concentration distribution in the sample reservoir prior to current measurement. These requirements are further described in the '069 and' 411 patents.

Column 4, lines 39 to 40 of the' 411 patent discloses the use of an initial incubation period of 15 to 90 seconds, preferably 20 to 45 seconds, for glucose testing. After the initial incubation period and application of the single excitation input signal, current measurements showing Cottrell decay may be recorded within 2 seconds to 30 seconds, or preferably 10 seconds to 20 seconds, after application of the input signal to the sensor strip. The requirement for a longer initial incubation period is also depicted in fig. 7 of the' 411 patent, where the sample is allowed to react (incubate) in the sensor strip for 160 seconds before the input signal is applied.

The longer incubation period required to fully convert the analyte into a measurable species provides: (1) time to allow hydration of the reagent layer containing the reagent; and (2) the time for converting the reagent to the analyte. For example, column 4, lines 36 to 44 of the' 411 patent describes a system having an incubation period long enough to allow the enzymatic reaction to reach completion. After this incubation period, during which the glucose analyte is completely converted to a measurable species, the instrument applies a known potential to the electrode to measure the resulting diffusion limited (i.e., Cottrell) current at a specific time during the decay of the resulting Cottrell current. Thus, conversion of the analyte to a measurable species is complete before a Cottrell decay is observed. It is also recognized in the' 411 patent that complete hydration of the reagent layer is a requirement for the Cottrell decay. The' 411 patent discloses that incomplete wetting of the reagent results in the system failing to decay following the Cottrell curve, which results in inaccurate analyte concentration values being obtained.

In addition to the extended incubation period, the Cottrell decay also requires that the measurable species in the sample have a substantially constant concentration profile with increasing distance from the electrode surface. Can be obtained by: (1) a relatively large sample volume; and/or (2) a relatively large distance between the end-face planar electrode or substantially planar electrode and the bottom surface of the sensor strip cover to achieve a substantially constant concentration profile. For example, column 8, line 40 of the' 069 patent describes a working electrode occupying a sample reservoir that provides a sample volume of 50 μ L, where the vertical distance between the working electrode and the lid is 500 μm to 2000 μm. In another example, unlike the closely spaced electrodes of the '102 patent, the distance between the working electrode and the counter electrode described in column 7, lines 62-66 of the' 411 patent must be at least 100 microns, and preferably greater than 100 microns.

Conventional analysis methods typically extend the time required to analyze the sample by requiring an incubation period, electrode distance, and sample reservoir volume sufficient to allow the system to have a Cottrell decay. Thus, there is an increasing need for improved biosensors; and in particular biosensors that more quickly determine the analyte concentration of a sample and do not rely on an estimate of the steady state current value. The systems, devices, and methods of the present invention overcome at least one of the disadvantages associated with conventional biosensors.

Disclosure of Invention

The present invention provides a biosensor system for determining an analyte concentration of a biological sample from an output signal having a transient decay. The output signal is not inversely proportional to the square root of time and therefore has a decay constant that is greater than or less than the decay constant of the Cottrell decay.

In one aspect, a method for determining an analyte concentration in a sample comprises: applying an input signal to the sample during an incubation period; generating an output signal having a transient decay in response to a redox reaction of a measurable species; and determining an analyte concentration from the output signal. The analyte may be glucose and the sample may be introduced into the sensor strip. The method may comprise transferring at least one electron from or to an analyte in the sample to form a measurable species, which may comprise at least one mediator.

The input signal may comprise at least two excitations separated by a relaxation, wherein the at least two excitations have a duration of 0.1 to 5 seconds and the duration of the relaxation is at least 0.1 seconds or at least 0.5 seconds. Each excitation and/or relaxation duration may be the same or different. The duration of one or more of the relaxations may be 0.1 to 3 seconds. The input signal may comprise at least three excitations and at least two relaxations. The input signal may comprise at least 2 duty cycles applied within 5 seconds.

For example, the incubation period can be 0.1 to 8 seconds, 0.1 to 6 seconds, or 0.5 to 4.75 seconds. The incubation period and application of the input signal may be completed in up to 12 seconds, up to 6 seconds, or up to 4 seconds. The transient decay may have a decay constant of-0.52 to-1 or-0.001 to-0.48. The transient decay may have a decay constant of at most-0.45 or at most-0.35. The output signal from which the analyte concentration is determined may comprise the current value recorded within 2 seconds of applying the input signal to the sample. The analyte concentration of the sample may be determined within at most 6 seconds, 3 seconds, or 1.5 seconds of applying the input signal.

The sample may be present in a reservoir defined by a sensor strip base and a bottom surface of a lid, the base being 20-200 microns from the bottom surface of the lid. The volume of the sample within the reservoir may be 0.25 microliters to 10 microliters or 0.25 microliters to 1.5 microliters. The reservoir may include at least one reagent layer having an average initial thickness of at most 20 microns, less than 14 microns, or at most 5 microns. When the input signal comprises at least two stimuli, the reservoir may comprise at least one reagent layer having an average initial thickness of at most 2 microns, at least one of the stimuli having a duration of at most 0.5 seconds. The reservoir may include at least one reagent layer comprising a separate diffusion barrier layer.

A reservoir height from the sensor strip base to a bottom of the lid may be up to 250 microns, a volume of sample within the reservoir may be up to 5 microliters, the reservoir may include at least one reagent layer having an average initial thickness of up to 20 microns, and the incubation period may be up to 12 seconds. A reservoir height from the sensor strip base to a bottom of the lid may be up to 150 microns, a volume of sample within the reservoir may be up to 3.5 microliters, the reservoir may include at least one reagent layer having an average initial thickness of less than 14 microns, and the incubation period may be up to 6 seconds. A reservoir height from the sensor strip base to a bottom of the lid may be at most 100 microns, a volume of sample within the reservoir may be at most 3 microliters, the reservoir may include at least one reagent layer having an average initial thickness of at most 2 microns, and the incubation period may be at most 2 seconds.

In another aspect, a method for determining an analyte concentration in a sample comprises: applying an input signal to the sample over an incubation period of up to 12 seconds; generating an output signal having a transient decay in response to a redox reaction of a measurable species; and determining an analyte concentration from the output signal.

In another aspect, a biosensor for determining an analyte concentration in a sample includes: a measurement device having a processor connected to the sensor interface; a sensor strip having a sample interface located on a base, the sensor interface in electrical communication with the sample interface, wherein the sample interface is adjacent to a reservoir formed by the base; wherein the processor instructs a charger to apply an input signal to the reservoir during an incubation period of at most 12 seconds; and wherein the processor determines the analyte concentration in the sample from the output signal having a transient decay in response to the redox reaction of the analyte in the sample.

The reservoir can include at least one working electrode in electrical communication with the charger, a reagent layer having a combined DBL/reagent layer on the working electrode, the combined DBL/reagent layer having an average initial thickness of from about 1 micron to about 20 microns. The combined DBL/reagent layer may have an average initial thickness of at most 1 micron.

In another aspect, a method for determining an analyte concentration in a sample comprises: applying an input signal to the sample over an incubation period of up to 12 seconds; generating a changing concentration profile of the measurable species in the sample reservoir; generating an output signal in response to a redox reaction of the measurable species; and determining an analyte concentration from the output signal.

In another aspect, a method for determining an analyte concentration in a sample comprises: introducing the sample into a sensor strip; applying an input signal to the sample over an incubation period of at most 8 seconds; generating an output signal having a transient decay in response to a redox reaction of a measurable species; and determining the analyte concentration from the transient decay of the output signal. The transient decay may be a decreasing current decay obtained within 0.5 seconds to 5 seconds or within about 0.5 seconds to about 3 seconds of applying the input signal to the sample.

Drawings

The invention can be better understood with reference to the following drawings and description. The components in the figures are not necessarily to scale, emphasis instead being placed upon illustrating the principles of the invention.

FIG. 1A is a perspective view of an assembled sensor strip.

Fig. 1B is a top view of a sensor strip with the cover removed.

FIG. 2A is an end view of the sensor strip of FIG. 1B.

Fig. 2B depicts a schematic of a biosensor system that determines the concentration of an analyte in a sample.

FIG. 3 shows a flow chart of an electrochemical method for determining the presence and/or concentration of an analyte in a sample.

FIG. 4A shows a sample reservoir bounded by a lower electrode surface and an upper lid.

FIG. 4B shows the incubation time t when elapsed before the input signal was applied₁To t₅According to the concentration profile formed by the sensor system.

FIG. 4C shows the relationship between the concentration of the measurable species in the reservoir and the rate of current decay.

FIG. 5 depicts the decay rate obtained from the working electrode after varying incubation periods for whole blood samples containing 50mg/dL, 100mg/dL, 200mg/dL, or 400mg/dL of glucose.

Fig. 6A-6C depict current distributions obtained from three sensor strips with different average initial thicknesses of the reaction layer over multiple initial incubation periods.

FIGS. 7A-7B depict the natural log current versus time for a whole blood sample including 100mg/dL or 300mg/dL glucose at 40% hematocrit obtained after a 6 second initial incubation period.

Fig. 8A to 8C are current decay profiles from an incubation period of 0.25 seconds, followed by a gated input signal with an excitation time of 0.5 seconds and a relaxation time of 0.25 seconds.

Fig. 8D is a calibration curve obtained by plotting the end point currents (p1, p2, p3) of the first three excitations obtained from the thin reagent layer sensor strip depicted in fig. 8A to 8C.

Fig. 8E is a calibration curve obtained by plotting the end point currents (p4, p5, p6) for excitations 4, 5, and 6 obtained from the sensor strip depicted in fig. 8A to 8C with a medium thickness reagent layer.

Detailed Description

Biosensor systems use electrochemical methods without the Cottrell decay constant to determine the analyte concentration of a biological sample. The biosensor system generates an output signal from a biological sample having a transient decay, wherein the output signal is not inversely related to the square root of time. The transient decay output of the biosensor system has a decay constant greater than or less than-0.5, and the system does not rely on an estimate of the steady state current value to determine the analyte concentration. Preferably, the transient decay upon which the analyte concentration is determined is decreasing.

The Cottrell decay is diffusion dependent, and does not exist unless the analyte has been completely converted to a measurable species and the measurable species in the sample reservoir is a substantially constant concentration profile prior to amperometric measurement. Relatively long incubation times and large sample volumes are required to obtain the Cottrell decay. Without these conditions, the output current will not be inversely related to the square root of time, and thus the biosensor will not exhibit the-0.5 decay constant required for the Cottrell decay. If the output current is not inversely related to the square root of time or if there is a decay constant other than-0.5 in the output signal, a biosensor designed to operate according to the Cottrell decay will provide an inaccurate analysis.

The biosensor system of the present invention operates using transient decay, where decay constants of less than or greater than-0.5 are observed. Transient and thus non-Cottrell decay constants can be obtained from a relatively short incubation period. The transient decay constant may also be derived from a relatively small sample reservoir volume, a relatively small distance between the electrode surface and the cover of the sensor strip, and/or a relatively short excitation related to the average initial thickness of the reagent layer.

To produce an output current with a transient decay or a transient decay constant greater or less than-0.5, the biosensor system may use an incubation period of 12 seconds or less, a reservoir volume of 5 μ L or less, a reservoir height of 200 μm or less, and/or an average initial thickness of the reagent layer of 20 μm or less. Preferred incubation periods for use in conjunction with a reservoir volume of 3.5 μ L or less, a reservoir height of 150 μm or less, and/or an average initial thickness of the reagent layer of 10 μm or less are at most 8 seconds, at most 6 seconds, or at most 4 seconds. Currently, a particularly preferred incubation period for use in conjunction with a sample tape sample volume of less than 3.0 μ L, a sample tape cap gap (cap-gap) height of less than 100 μm, and/or an average initial thickness of the reagent layer of less than 2 μm is at most 2 seconds or at most 1 second. Other incubation periods, reservoir volumes, reservoir heights, and reagent layer thicknesses may be used.

Fig. 1A and 1B depict a sensor strip 100 that may be used in a biosensor system. FIG. 1A is a perspective view of an assembled sensor strip 100 including a sensor base 110, at least a portion of the sensor base 110 being covered by a cover 120, the cover including a vent opening 130, a sample coverage area 140, and an input end opening 150. A partially enclosed sample reservoir 160 (capillary gap or cap gap) is formed between the base 110 and the lid 120. Other sensor strip designs, such as those described in U.S. Pat. Nos. 5,120,420 and 5,798,031, may also be used. Although a particular configuration is illustrated in fig. 1A-1B, sensor strip 100 may have other configurations, including configurations with additional components.

The height of the reservoir 160 located between the sensor base 110 and the cover 120 may be 20 to 250 micrometers (μm), more preferably 50 to 150 micrometers. The volume of the reservoir 160 may be 0.25 to 10. mu.L, preferably 0.8 to 4. mu.L, more preferably 0.5 to 1.5. mu.L. Other heights and volumes may be used.

A liquid sample for analysis may be transferred into the reservoir 160 by introducing a liquid into the opening 150. The liquid fills the reservoir 160 while expelling the previously contained air through the vent 130. The reservoir 160 may contain a composition (not shown) that helps to retain the liquid sample in the reservoir. Examples of such compositions include water-swellable polymers such as carboxymethylcellulose and polyethylene glycol; and porous polymer matrices such as dextran and polyacrylamide.

FIG. 1B depicts a top view of the sensor strip 100 with the cover 120 removed. Conductors 170 and 180 may be connected from opening 150 to working electrode 175 and counter electrode 185, respectively, under dielectric layer 190. The sensor strip 100 may include more than one working electrode. The working electrode 175 and the counter electrode 185 can be in substantially the same plane. The electrodes may be in another orientation. The dielectric layer 190 may partially cover the electrodes 175, 185 and may be made of any suitable dielectric substance, such as an insulating polymer. Although a particular electrode configuration is shown, the electrode may have other configurations, including configurations with additional components.

The counter electrode 185 may support electrochemical activity at the working electrode 175 of the sensor strip 100. A potential is provided to the sensor system that supports electrochemical activity at the working electrode 175 by forming the counter electrode 185 from an inert material, such as carbon, and including a soluble redox species, such as ferricyanide, within the reservoir 160. The potential at counter electrode 185 can be a reference potential achieved by forming counter electrode 185 with a redox couple, such as Ag/AgCl, to provide a combined reference-counter electrode. A redox couple includes two conjugates of chemical species having different oxidation numbers. Reduction of species with higher oxidation numbers will produce species with lower oxidation numbers. Alternatively, oxidation of species with lower oxidation numbers will produce species with higher oxidation numbers. The sensor strip 100 may be provided with a third conductor and electrodes to provide a reference potential to the sensor system.

Working electrode 175 can be spaced greater than 200 μm or 250 μm from counter electrode 185. Working electrode 175 and counter electrode 185 may be spaced less than 200 μm apart. Working electrode 175 can be spaced apart from counter electrode 185 at other distances.

FIG. 2A depicts an end view of the sensor strip 100 depicted in FIG. 1B, showing the layered structure of the working electrode 175 and the counter electrode 185 present within the reservoir 160. Conductors 170 and 180 may be located on base 110. Other substances may be present between the conductors 170, 180 and the base 110, and thus these conductors may or may not be in physical contact with the base. A portion of the conductor may pass through a portion of the base. Surface conductor layers 270 and 280 may optionally be deposited on conductors 170 and 180, respectively. Other substances may be present between the surface conductor layers 270, 280 and the conductors 170, 180, and thus these surface conductors may or may not be in physical contact with the conductors. A portion of the surface conductor may pass through a portion of the conductor. The surface conductor layers 270, 280 may be made of the same substance or of different substances.

The substance used to form the conductors 170, 180 and surface conductor layers 270, 280 includes any electrical conductor. The conductors 170, 180 preferably comprise a thin layer of a metal or metal paste such as gold, silver, platinum, palladium, copper or tungsten. The surface conductor layers 270, 280 preferably comprise carbon, gold, platinum, palladium, or combinations thereof. Preferred electrical conductors are non-ionizing such that no net oxidation or net reduction of species occurs during sample analysis. Thus, if no surface conductor layer is present on the conductor, the conductor is preferably made of a non-ionizing substance, such as carbon, gold, platinum, palladium, or a combination thereof.

The surface conductor material may be deposited on the conductors 170, 180 by any conventional means compatible with sensor strip operation, including foil deposition, chemical vapor deposition, slurry deposition, and the like. In the case of slurry deposition, the conductor substance may be applied to the conductors 170, 180 in the form of an ink, as described in U.S. Pat. No. 5,798,031.

Reagent layers 275 and 285 may be deposited on conductors 170 and 180, respectively. These layers are formed from at least one reagent composition that may include a binder. The binder is preferably an at least partially water soluble polymeric substance. The adhesive may form a gel or gel-like substance when hydrated. The adhesive, when hydrated, may bind the agent to form a gel or gel-like substance. The gel or gel-like substance may inhibit and/or filter red blood cells from reaching surface conductor 270 and/or conductor 170.

Partially water soluble polymeric materials suitable for use as adhesives may include: polyethylene oxide (PEO), carboxymethylcellulose (CMC), polyvinyl alcohol (PVA), hydroxyethylidene cellulose (HEC), hydroxypropyl cellulose (HPC), methylcellulose, ethylcellulose, ethylhydroxyethyl cellulose, carboxymethylethylcellulose, polyvinylpyrrolidone (PVP), polyamino acids (e.g., polylysine), polystyrene sulfonate, gelatin, acrylic acid, methacrylic acid, starch, maleic anhydride, salts thereof, derivatives thereof, and combinations thereof. Currently, among the above binder substances, PEO, PVA, CMC and HEC are preferable, with CMC being more preferable.

In addition to the binder, the reagent layers 275 and 285 may include the same or different reagents. When the same reagent is included, reagent layers 275 and 285 may be the same layer. In one aspect, the reagent present in the first layer 275 can be selected for use with the working electrode 175, while the reagent present in the second layer 285 can be selected for use with the counter electrode 185. For example, the reagent in layer 285 may facilitate the flow of electrons between the sample and conductor 180. Similarly, the reagents in layer 275 may facilitate the reaction of the analyte.

The reagent layer 275 may include an enzyme system specific to the analyte that may enhance the specificity of the sensor system for the analyte, particularly in complex biological samples. The enzyme system may include one or more enzymes, cofactors and/or other moieties that participate in the redox reaction of the analyte. For example, alcohol oxidase can be used to provide a sensor strip that is sensitive to the presence of alcohol in a sample. The system may be adapted to measure blood alcohol concentration. In another example, glucose dehydrogenase or glucose oxidase can be used to provide a sensor strip that is sensitive to the presence of glucose in a sample. For example, the system may be used to measure blood glucose concentrations in patients known or suspected to have diabetes.

Enzymes used in the enzyme system include: alcohol dehydrogenase, lactate dehydrogenase, beta-hydroxybutyrate dehydrogenase, glucose-6-phosphate dehydrogenase, glucose dehydrogenase, formaldehyde dehydrogenase, malate dehydrogenase, and 3-hydroxysteroid dehydrogenase. Preferred enzyme systems may be oxygen independent and therefore substantially not oxidized by oxygen.

One family of oxygen-independent enzymes used in glucose sensor strips is Glucose Dehydrogenase (GDH). With different coenzymes or cofactors, GDH can be mediated by different mediators in different ways. Depending on the association with GDH, cofactors such as Flavin Adenine Dinucleotide (FAD) can be fastened by the host enzyme, as is the case with FAD-GDH; or co-factors such as pyrroloquinoline quinone (PQQ)The molecule may be covalently linked to the host enzyme, as is the case for PQQ-GDH. The cofactor in each of these enzyme systems may be immobilized by the master enzyme, or the coenzyme and the dehyrocoenzyme may be reconstituted prior to addition of the enzyme system to the reagent composition. A coenzyme may also be added independently to the primary enzyme in the reagent composition to assist the catalytic function of the primary enzyme, for example in nicotinamide adenine dinucleotide NAD/NADH⁺Or nicotinamide adenine dinucleotide phosphate NADP/NADPH⁺In the case of (1).

Reagent layer 275 may also include a mediator to more effectively communicate the results of the analyte redox reaction to surface conductor 270 and/or conductor 170. Mediators can be divided into two classes based on electrochemical activity. The single electron transfer mediator is capable of taking one additional electron during the electrochemical reaction. Examples of the one-electron transfer mediator include compounds such as 1, 1' dimethylferrocene, ferrocyanide and ferricyanide, and hexaamineruthenium (III). The two electron transfer mediator is capable of taking two additional electrons.

Two electron mediators include organic quinones and hydroquinones such as phenanthroline quinone; phenothiazine and phenoxazine derivatives; 3- (phenylamino) -3H-phenoxazine; phenothiazine; and 7-hydroxy-9, 9-dimethyl-9H-acridin-2-one and its derivatives. Examples of other two-electron mediators include electroactive organic molecules as described in U.S. Pat. Nos. 5,393,615, 5,498,542, and 5,520,786, which are incorporated herein by reference. Other electroactive organic molecules include organic molecules that are free of metals and that are capable of undergoing redox reactions. The electroactive organic molecule may act as a redox species and/or mediator. Examples of electroactive organic molecules include coenzyme pyrroloquinoline quinone (PQQ), benzoquinones and naphthoquinones, N-oxides, nitroso compounds, hydroxylamines, hydroxyquinolines, flavins, phenazines, phenothiazines, indophenols, and indamines.

Preferred two-electron transfer mediators include 3-phenylimino-3H-phenothiazine (PIPT) and 3-phenylimino-3H-phenoxazine (PIPO). More preferred dual electron mediators include carboxylic acids or salts of phenothiazine derivatives, such as ammonium salts. Currently, particularly preferred two electron mediators include (E) -2- (3H-phenothiazin-3-ylideneamino) benzene-1, 4-disulfonic acid, (E) -5- (3H-phenothiazin-3-ylideneamino) isophthalic acid, (E) -3- (3H-phenothiazin-3-ylideneamino) -5-carboxybenzoic acid ammonium salt, and combinations thereof. Preferred dual electron mediators have a redox potential that is at least 100mV lower, more preferably at least 150mV lower than ferricyanide.

The reagent layers 275, 285 may be deposited by any convenient means such as printing, liquid deposition, or ink jet deposition. In one aspect, the layers are deposited by printing. The angle of the printing blade may adversely affect the initial thickness of the reagent layer, all other factors being equal. For example, the layer may have an initial thickness of about 10 μm when the blade is moved at an angle of about 80 ° to the base 110. Similarly, when a blade angle of about 62 ° to the base 110 is used, a layer thicker than 30 μm may be produced. Thus, a smaller doctor angle may provide a thicker reagent layer. In addition to the doctor blade angle, other factors such as the viscosity of the reagent composition and the sieve size and emulsion combination may also affect the resulting thickness of the reagent layers 275, 285.

When a thinner reagent layer is preferred, deposition methods other than printing may be used, such as micropipette, ink jet, or pin deposition. These deposition methods typically produce a layer of dry reagent of micron or sub-micron thickness, such as 1-2 μm. For example, pin deposition methods may provide an average initial thickness of about 1 μm for the reagent layer. The reagent layer thickness produced by pin deposition methods, for example, can be controlled by the amount of polymer included in the reagent composition, with higher polymer content providing a thicker reagent layer. Thinner reagent layers may require shorter excitation durations than thicker reagent layers to maintain desired measurement performance and/or to substantially measure analytes within the Diffusion Barrier (DBL).

The working electrode 175 can include a DBL integral with the reagent layer 275 or as a separate layer 290, as shown in fig. 2A. Thus, the DBL may be formed on the conductor in a combined reagent/DBL form, as a separate layer on the conductor, or as a separate layer on the reagent layer. When the working electrode 175 includes a separate DBL 290, the reagent layer 275 may or may not be located on the DBL 290. Rather, the reagent layer 275 may be located on any portion of the sensor strip 100 that dissolves the reagent in the sample. For example, the reagent layer 175 can be located on the base 110 or on the lid 120.

The DBL provides a porous space having an interior volume in which measurable species can be located and also filters red blood cells from the conductive surface. The pores of the DBL may be selected such that measurable species may diffuse into the DBL, substantially excluding physically larger sample components (e.g., red blood cells). While conventional sensor strips have used various substances to filter red blood cells from the surface of the working electrode, the DBL provides an internal porous space to hold and separate a portion of the measurable substance from the sample.

When the reagent layer 275 includes a water-soluble binder, any portion of the binder that is not soluble in the sample before the stimulus is applied can act as an integral DBL. The average initial thickness of the combined DBL/reagent layer is preferably less than 20 μm or 10 μm and more preferably less than 5 μm. The desired average initial thickness of the combined DBL/reagent layer can be selected for a particular excitation length based on the diffusion rate of the measurable species from the DBL to the conductor surface (e.g., the surface of conductor 170 or the surface of surface conductor 270 of fig. 2A) becoming relatively constant. When combined with an actuation duration of 0.25 seconds or less, the combined DBL/reagent layer may have an average initial thickness of 2 μm, 1 μm, or less.

The independent DBL 290 may include any substance that provides the desired porosity while partially or slowly dissolving in the sample. The individual DBL 290 may include an agent that does not include an agent binding substance. The individual DBLs 290 may have an average initial thickness of 1 to 15 μm, more preferably 2 to 5 μm.

Fig. 2B depicts a schematic diagram of a biosensor system 200 that determines an analyte concentration in a sample, such as a biological fluid. The biosensor system 200 includes a measurement device 202 that performs an analytical method and a sensor strip 204. For example, sensor strip 204 may be an electrochemical sensor strip as depicted in fig. 1A, 1B, and 2A. The measurement device 202 may be implemented as a desktop device, a portable or handheld device, or the like. The measurement device 202 and the sensor strip 204 may perform electrochemical analysis, optical analysis, a combination thereof, or the like. The biosensor system 200 may determine analyte concentrations, including analyte concentrations of alcohol, glucose, uric acid, lactate, cholesterol, bilirubin, etc., in a biological sample. Although a particular configuration of biosensor 200 is shown, it may have other configurations, including configurations with additional components.

The sensor strip 204 has a base 206 forming a sample reservoir 208 and a channel 210 having an opening 212. Referring to fig. 1A, the channel 210 may be integrally formed with the reservoir 208. The reservoir 208 and channel 210 may be covered by a lid having a vent. The reservoir 208 defines a partially enclosed volume (cap gap). The reservoir 208 may contain components such as a water-swellable polymer or a porous polymer matrix to assist in retaining the liquid sample. Reagents may be deposited in the reservoir 208 and/or the channel 210. The reagent composition may include one or more enzymes, binders, mediators, and the like. The reagent may include a chemical indicator for the optical system. The sensor strip 204 may have other configurations.

The sensor strip 204 may also have a sample interface 214. In an electrochemical system, the sample interface 214 has conductors that connect to at least two electrodes, such as a working electrode and a counter electrode. The electrodes may be deposited on the surface of the substrate 206 forming the reservoir 208. The sample interface 214 may have other electrodes and/or conductors.

The measurement device 202 includes circuitry 216 connected to a sensor interface 218 and a display 220. The circuit 216 may include a processor 222 connected to a signal generator 224, an optional temperature sensor 226, and a storage medium 228. The circuit 216 may have other configurations, including configurations with additional components.

The signal generator 224 provides an electrical input signal to the sensor interface 218 in response to the processor 222. In an optical system, the detector and light source in sensor interface 218 may be operated or controlled using electrical input signals. In an electrochemical system, the sensor interface 218 may transmit an electrical input signal to the sample interface 214 to apply the electrical input signal to the reservoir 208 and thereby to the sample.

The electrical input signal may be a potential or a current and may be constant, variable, or a combination thereof, such as when an AC signal is applied by a DC signal offset. The electrical input signal may be applied as a single pulse or in multiple pulses, sequences or cycles. The signal generator 224 may also record the output signal from the sensor interface 218 as a generator-recorder.

The storage medium 228 may be a magnetic, optical, or semiconductor memory, other computer readable storage device, or the like. The storage medium 228 may be a fixed storage device or a removable storage device such as a memory card.

The processor 222 performs analyte analysis and data processing using computer readable software code and data stored in the storage medium 228. The processor 222 may initiate analyte analysis in response to the presence of the sensor strip 204 at the sensor interface 218, application of a sample to the sensor strip 204, user input, and the like. The processor 222 instructs the signal generator 224 to provide the electrical input signal to the sensor interface 218. The processor 222 may receive the sample temperature from the temperature sensor 226 (if so equipped).

The processor 222 receives the output signal from the sensor interface 218. The output signal is generated in response to a redox reaction of an analyte in the sample. An optical system, an electrochemical system, or the like may be used to generate the output signal. Processor 222 may determine the concentration of the analyte in the sample from the one or more output signals using a correlation equation. The results of the analyte analysis are output to the display 220 and may be stored in the storage medium 228.

The correlation equation relating analyte concentration to output signal may be represented graphically, mathematically, a combination thereof, or the like. The correlation equation may be represented by a Program Number Assignment (PNA) table, another look-up table, etc. stored in the storage medium 228. The instructions related to the implementation of the analysis may be provided by computer readable software program code stored in storage medium 228. The code may be object code or any other code that describes or controls the functions described herein. The data from the analyte analysis may be subjected to one or more data processing in processor 222, including determination of decay rate, K-constant, slope, intercept and/or sample temperature.

In an electrochemical system, the sensor interface 218 is in electrical or optical communication with the sample interface 214. Electrical communication includes transferring input and/or output signals between contacts in sensor interface 218 and conductors in sample interface 214. For example, electrical communication may be effected wirelessly or by physical contact. The sensor interface 218 transmits the electrical input signal from the signal generator 224 to the connector in the sample interface 214 via the contact points. The sensor interface 218 also transmits output signals from the sample to the processor 222 and/or the signal generator 224 via the contact points.

Optical communication includes transmitting light between an optical inlet in the sample interface 202 and a detector in the sensor interface 208. Optical communication also includes transmitting light between an optical inlet in the sample interface 202 and a light source in the sensor interface 208.

The display 220 may be of an analog or digital type. The display 220 may be an LCD, LED or vacuum fluorescent display suitable for displaying numerical readings.

In use, a liquid sample for analysis is transferred into the reservoir 208 by introducing the liquid sample into the opening 212. The liquid sample flows through the channel 210 and into the reservoir 208 while venting the previously contained air. The liquid sample chemically reacts with reagents deposited in the channel 210 and/or reservoir 208. The processor 222 instructs the signal generator 224 to provide an input signal to the sensor interface 218. In the optical system, the sensor interface 218 operates the detector and the light source in response to the input signal. In an electrochemical system, the sensor interface 218 provides the input signal to the sample via the sample interface 214. Processor 222 receives an output signal generated in response to a redox reaction of an analyte in a sample. Processor 222 uses one or more correlation equations to determine the analyte concentration of the sample. The determined analyte concentration may be displayed and/or stored for future reference.

Fig. 3 shows a flow diagram of an electrochemical assay 300 for determining the presence and optionally concentration of an analyte 322 in a sample 312. At 310, a sample 312 is introduced into a sensor strip 314 (e.g., the sensor strip shown in fig. 1A-1B and fig. 2A). A reagent layer, such as 275 and/or 285 depicted in fig. 2A, begins to dissolve in the sample 312, thereby allowing the reaction to proceed.

In 315, an initial incubation period 317 allows the reagent to react with the sample 312 before the input signal is applied. Preferably, incubation period 317 may be 0.1 to 10 seconds, more preferably 0.1 to 8 seconds or 0.5 to 4 seconds. Currently, 0.1 seconds to 1 second is more preferable for the incubation period 317. Other incubation periods may be used.

During incubation period 317, in 320, a portion of analyte 322 present in sample 312 is chemically or biochemically oxidized or reduced by a redox reaction to form measurable species 332. Measurable species 332 can be an oxidized or reduced analyte 322 or mediator. Upon oxidation or reduction, electrons may be transferred to analyte 322 or from analyte 322 and to or from measurable species 332 in 330. For example, the mediator may be reduced via oxidation of the analyte 322 to form the measurable species 332. Preferably, measurable species 332 formed during incubation period 317 is not electrochemically excited during incubation period 317.

At 340, measurable species 332 is electrochemically activated (oxidized or reduced). In this manner, electrons are selectively transferred between analyte 322 and the working electrode of sensor strip 314. The duration of the excitation 340 may be 0.1 to 5 seconds or 0.1 to 1 second. The excitation 340 may be repeated.

At 350, the current generated during the excitation 340 may be recorded as a function of time. If multiple excitations 340 are applied to the sensor band 314, one or more currents due to the excitations 340 may be recorded at 350. The current may be recorded by a measuring device.

At 360, the sample undergoes relaxation. Preferably, no current is recorded during the relaxation 360. When multiple excitations are applied, relaxation 360 may occur after each excitation 340. During relaxation 360, the current present during excitation 340 is substantially reduced by at least half, preferably by an order of magnitude, and more preferably to zero. Preferably, the zero current condition is achieved by an open circuit or other method known to those skilled in the art to provide substantially zero current flow. The measurement device may break the electrical circuit through the sensor strip 314 to provide an open circuit. If a zero current state is provided, the relaxation 360 may be considered an intermittent incubation period.

The duration of the relaxation 360 may be at least 0.1 seconds or at least 0.5 seconds. The duration of the relaxation 360 may be 0.1 to 3 seconds, 0.2 to 2 seconds, or 0.5 to 1 second. Other relaxation durations may be used.

At 370, one or more of the current and time values recorded at 350 may be analyzed to determine the presence and/or concentration of analyte 322 in sample 312. Preferably, the analyte concentration is determined from current measurements taken within 2 seconds or 1 second of the start of the initially applied excitation. More preferably, a plurality of shorter excitations are combined with current measurements taken within 2 seconds, 1 second, or less of the beginning of the initially applied input signal to determine the analyte concentration of the sample. The recorded current and time values may be correlated to the concentration of analyte 322 in sample 312 using one or more correlation equations.

The excitation 340 and relaxation 360 constitute a single duty cycle. Preferably, the input signal applied to sensor strip 314 comprises at least 2, 4, or 6 duty cycles applied over an independently selected period of 3 seconds, 5 seconds, 7 seconds, or 9 seconds. Thus, the total time required for the excitation 340 and relaxation 360 portions of the electrochemical analysis 300 may be at most 3 seconds, at most 5 seconds, at most 7 seconds, or at most 9 seconds from the initial application of the input signal. The duty cycle may be applied over a period of 1 second to 3 seconds. 2 to 6 duty cycles may be applied in a period of 8 seconds or less. 2 to 4 duty cycles can be applied in 3 to 6 seconds. Other time periods may be used.

The duty cycle may be repeated continuously for continuous monitoring (e.g., as may be used in conjunction with implantable or partially implantable sensors). The energy required to operate the system may be reduced and the useful life of the system may be extended relative to a method without relaxation. Further, the application of multiple duty cycles may be separated by a longer period of time, such as 5 minutes or more.

Amperometric sensor systems apply a potential (voltage) to the electrodes to excite the measurable species while monitoring the current (amperage). Conventional amperometric sensor systems may maintain the excitation potential while constantly measuring the current for, for example, 5 to 10 seconds. The input signal used in electrochemical analysis 300 may replace a continuous longer duration excitation with a plurality of relatively shorter duration excitations and relaxations as compared to conventional methods. A more detailed description of a plurality of excitation and relaxation or "Gated" pulse sequences applied as input signals can be found in WO 2007/013915 entitled "Gated Amperometry", filed on 19.7.2006.

Transient or non-Cottrell current decay may result when the shorter initial incubation time and/or gated input signal of the present invention is used. Determining the concentration of analyte 322 in sample 312 independent of the-0.5 Cottrell decay constant enables electrochemical analysis 300 to be completed using transient decay within 8 seconds or less, within 4 seconds or less, or more preferably within 3 seconds or less. Electrochemical analysis 300 can be completed in less than 2 seconds. Electrochemical analysis 300 can be completed in about 0.5 seconds to about 3 seconds. Other time periods may be used to complete the electrochemical analysis 300 using transient decay.

Fig. 4A shows a sample reservoir 400 bounded by a lower electrode surface 402 and an upper lid 403. The virtual upper limit 105 of the reagent layer is also shown. Thus, the region between the electrode surface 402 and the virtual upper boundary 405 represents the sample contained in the reagent layer. Similarly, the area between the virtual upper limit 405 and the upper cover 403 represents the sample above the reagent layer. The x-axis represents distance from the electrode surface, while the y-axis represents the sample concentration of the measurable species produced by the redox reaction of the analyte. The figure omits the role of the analyte separating between the DBL and the liquid sample located in the rest of the reservoir 400.

Concentration profile 410 represents what would be observed immediately after the sample was introduced into the sensor strip, while concentration profile 420 represents what would be observed after a relatively long incubation period. Concentration profile 410 represents an instantaneous condition, while concentration profile 420 represents a Cottrell condition. There may be multiple transient states between the transient concentration profile 410 and the Cottrell concentration profile 420.

FIG. 4B shows the incubation time t when elapsed before the input signal was applied to the electrodes₁To t₅The formation of different concentration profiles. T representing an incubation period of 15 seconds to 30 seconds₅The concentration profile at (a) depicts a substantially constant concentration profile of the measurable species throughout the sample that will provide a Cottrell decay having a decay constant of-0.5. Thus, t₅The area under the line and the associated concentration of the measurable species does not change significantly until a relatively large distance from the electrode surface 402.

And t₅Line comparison, t₄The line has an incubation period of 1 to 12 seconds and a changing concentration profile of the measurable species in the sample. t is t₄The line has a slower transient decay constant of-0.30 (1 second) to-0.48 (12 seconds). Thus, t₄The measurable species concentration in the area and lower portion under the line varies significantly from the electrode surface 402 of the reservoir 400 to the upper cover 403 and thus is variable.

With t₃Further reducing the incubation period to 0.4 to 1 second or at t₂Reduced to 0.1-0.3 seconds, the transient decay constant can be respectively aimed at t₃In the range of-0.25 to-0.3 and for t₂In the range of-0.15 to-0.25. T represents an incubation period of 0.01 to 0.1 seconds₁The decay may have a transient decay constant below-0.15. With the cultivation period from t₄To t₁As a result, the area under these lines and the concentration of the associated measurable species between the electrode surface 402 of the reservoir 400 and the upper cover 403 become increasingly lessThe same is true.

By having a lower concentration of measurable species on the electrode surface 402 than in the remainder of the reservoir 400 (such as t of fig. 4B₁To t₄As indicated by the changing concentration profile), the rate of current decay may be slower than the-0.5 decay constant required for Cottrell decay. This slower decay may be attributed to the large concentration of the measurable species that is farther from the electrode surface 402 reaching the electrode surface faster than if the measurable species were uniformly distributed within the sample reservoir 400. Similarly, faster decay rates can be obtained when there is a higher concentration of measurable species on the electrode surface 402 than in the rest of the sample reservoir 400.

FIG. 4C shows the relationship between the concentration of the measurable species in the reservoir 400 and the current decay constant. The concentration profiles 430 and 440 of the measurable species have decay rates slower and faster than 420, respectively, which corresponds to a-0.5 Cottrell decay constant. For a concentration profile 430 having a decay constant less than-0.5 Cottrell decay constant, such as-0.3, the rate of current decay will be slower than that observed for the Cottrell system. Similarly, for a concentration profile 440 having a decay constant greater than-0.5 Cottrell decay constant (such as-0.7), the rate of current decay will be faster than that observed for the Cottrell system. Thus, the transient decay constants 430, 440 reflect different concentration distributions of the measurable species in the reservoir 400, as compared to the-0.5 Cottrell decay constant represented by 420.

When a longer incubation period is used to produce the Cottrell decay, the amount of measurable species produced during the measurement excitation is small compared to the amount of measurable species produced during a previous incubation period. Thus, rather than concentration profile 420 representing complete redox conversion of the analyte to a measurable species prior to application of the input signal, concentration profiles 430, 440 represent incomplete conversion. In addition, any change in the rate at which the measurable species diffuses to the electrode by convection or other means is also small relative to the amount of measurable species produced during the incubation period. Thus, the effect of changing the-0.5 Cottrell decay constant is essentially offset by the longer incubation period.

In contrast, when shorter incubation periods are used, such as 12 seconds, 10 seconds, and shorter, any change in the amount of measurable species produced during measurement excitation and the diffusion rate from methods other than diffusion can provide an actual decay rate that is slower than the-0.5 Cottrell value. This decay process can be described by the following normalized current equation (2)):

f(t)＝t^-a+b+c (2)，

where a is the fraction of the decay constant from the measurable species formed during the incubation period, b is the fraction of the decay constant from the measurable species formed during the measurement excitation, and c is the fraction of the decay constant caused by the change in the concentration profile of the measurable species in the sample reservoir. Negative values of b and c result in an increase in the measured concentration of the measurable species, while positive values of b and c result in a decrease in the measured concentration of the measurable species. Thus, if a or b is non-zero, a deviation from the decay value of a will result. Since the Cottrell decay is provided at a-0.5 value for a, the significant contribution of b or c provides the transient decay constant. In equation (2), term a controls the decay constant obtained from concentration profile 420, while term b will significantly affect the decay constant obtained from concentration profiles 430 and 440, where the input signal is applied before the redox conversion of the analyte is complete.

Equation (2) establishes: the decay constant of the system may vary over time in response to which of these fundamental factors affect the current decay when measured. For example, a longer incubation period increases a and decreases b because the more analyte that is converted to a measurable species during the incubation period, the less analyte remains in the sample to be converted to a measurable species during the excitation period.

The redox conversion of the analyte to the measurable species occurs in the hydrated reagent layer. Since a thicker reagent layer will take longer to hydrate, if the input signal is applied before the reagent layer hydrates, the thicker reagent layer will provide an increase in b relative to a. Due to the effect on the decay constant (term b of equation (2)) of the measurable species formed during the measurement excitation, Cottrell decay is not observed before the reagent layer hydrates. This is recognized in column 4, lines 58 to 59 of the' 069 patent, which reveals that incomplete wetting of the reagent results in the system failing to decay following the Cottrell curve, resulting in inaccurate analyte concentration values being obtained. Thus, the transient decay constant may be obtained from a partially hydrated reagent layer resulting from a relatively short initial incubation period.

A sensor strip reservoir comprising a distribution of measurable species at a substantially constant concentration may reduce any effect on the decay constant attributed to c. If the excitation duration is too long for the sample volume, the c term can also affect the decay constant, resulting in a rapid decrease in the measurable species concentration with increasing distance from the electrode surface. Using a shorter excitation or multiple shorter excitations in combination with one or more relaxations may help to reduce the influence of the c term on the decay constant.

For example, the' 069 patent describes a system that provides a-0.5 Cottrell decay constant when a 160 second initial incubation period is combined with a 50 μ L sample reservoir. For this system, if the incubation period is sufficiently shortened, the b term of equation (2) will increase, thereby providing a non-Cottrell decay. Similarly, if the reservoir volume is sufficiently reduced, non-Cottrell decay will result due to the increase in the c term of equation (2).

FIG. 5 depicts decay constants obtained from sensor strips having a reservoir volume of about 3.5 μ L and an electrode-to-lid distance of about 250 μm after varying incubation periods for whole blood samples containing glucose of 50mg/dL, 100mg/dL, 200mg/dL, or 400 mg/dL. The decay rate increases with increasing incubation time; however, a Cottrell decay constant of-0.5 was not obtained during the 6 second incubation period. Thus, the system provides transient decay under these conditions.

Table I below provides the decay constants for the 1 second to 6 second incubation periods of fig. 5 and provides the predicted constants for the 10 second to 15 second incubation periods. Also provides a predicted decay constant for an extended 20 second incubation period.

TABLE I

Input signal	Period of cultivation	50mg/dL	100mg/dL	200mg/dL	400mg/dL
						4-1-1	1	-0.2479	-0.23823	-0.2119	-0.17947
4-2-1	2	-0.337	-0.30593	-0.282	-0.2631
						4-4-1	4	-0.37417	-0.34993	-0.3442	-0.32837
4-5-1	5	-0.3877	-0.3734	-0.3549	-0.35283
						4-6-1	6	-0.3979	-0.38273	-0.373	-0.36483
Prediction of	10	-0.44596	-0.42622	-0.42066	-0.42275
						Prediction of	15	-0.4786	-0.45853	-0.45679	-0.46475
Prediction of	20	-0.50176	-0.48146	-0.48242	-0.49456

In each case, the input signal comprised an initial excitation of 4 seconds, followed by an intermittent incubation period of open circuit type with varying duration and a measured excitation for a period of 1 second for recording the current. The sensor system did not reach the Cottrell decay condition during any of the incubation periods of 1-6 seconds. Even at lower glucose concentrations of 50mg/dL, the sensor system would not be able to predict the Cottrell decay condition within 12 seconds. Preferred transient decay constants are-0.001 to-0.48 and-0.52 to-1. More preferred transient decay constants are at most-0.45, at most 0.35, and at most-0.3. Other transient decay constants may be used.

Fig. 6A-6C depict current distributions obtained from three sensor strips with different average initial thicknesses of the reaction layer at initial incubation periods of 0.125 seconds, 0.5 seconds, 1 second, 2 seconds, 4 seconds, and 6 seconds. The sample reservoir for each sensor strip was about 1 μ L. FIG. 6A is a graph obtained from a plurality of sensor strips having reaction layers with an average initial thickness ("thickness") of about 15 μm to about 20 μm. The curves of fig. 6B and 6C were obtained from sensor strips with reaction layers having average initial thicknesses of 10 μm to 15 μm ("medium") and 1 μm to 2 μm ("thin"), respectively. Other thicknesses may be used.

These plots establish the relationship of incubation time, reagent layer thickness and associated layer hydration rate. A thicker reagent layer requires a longer time to hydrate the reagent layer, and the longer the time required to hydrate the reagent layer, the longer the time before the current decays to reach the point of continuous decrease. The current value obtained from the decreasing transient decay may preferably be used to correlate with the analyte concentration of the sample.

For the thick layer sensor strip of fig. 6A, a continuously decreasing current decay was obtained after an incubation period of more than 4 seconds. However, for incubation periods below about 2 seconds, no continuously decreasing current decay is obtained for thick layer sensor strips until the input signal is applied for more than about 2 seconds.

For the medium thickness reagent layer of the sensor strip of fig. 6B, a continuously decreasing current decay is obtained after an incubation period of about 2 seconds or more. For incubation periods below about 1 second, input signals above about 2 seconds provide a continuously decreasing current decay.

For the thin reagent layer of the sensor strip of fig. 6C, a continuously decreasing current decay is obtained after an incubation period of more than about 1 second. For incubation periods below about 0.5 seconds, input signals above about 1 second provide a continuously decreasing current decay. Thus, thinner reagent layers may be combined with shorter incubation periods to provide shorter overall analysis times, while thicker reagent layers may require longer duration incubation periods and/or input signals.

FIGS. 7A-7B depict the natural log current versus time for a whole blood sample including 100mg/dL or 300mg/dL glucose at 40% hematocrit obtained after a 6 second initial incubation period. The sample reservoir volume and initial average thickness of the reagent layer are the same as in fig. 6A-6C above. These curves are generated from current values obtained during 5 seconds prior to the 10 second excitation, where the a term of equation (2) dominates the decay constant. Each of the observed decay constants (slope of ln (current, nA) versus ln (time, seconds) curves) differs from the-0.5 Cottrell decay constant, with a transient decay constant in the range of about-0.35 to about-0.45. Thus, even at the longest initial incubation period of 6 seconds, Cottrell decay is not observed.

Fig. 8A-8C are decay profiles of a gated input signal from an initial incubation period of 0.25 seconds, followed by an excitation of 0.5 seconds and a relaxation of 0.25 seconds to provide a duty cycle duration of 0.75 seconds. Whole blood samples including 50mg/dL, 100mg/dL, or 400mg/dL glucose at 40% hematocrit were analyzed using a medium reagent layer sensor strip and a thin reagent layer sensor strip having a sample reservoir volume of about 1 μ L. For a thin reagent layer, a continuously decreasing current decay is obtained within 0.75 seconds (and thus during the first excitation period) that can be correlated to a 50mg/dL analyte concentration in the sample. For thicker intermediate reagent layers, a continuously decreasing current decay is obtained within 3 seconds (and thus during the third excitation period).

Fig. 8D is a calibration curve obtained by plotting the end currents (p1, p2, p3) of the first three excitations obtained from the thin reagent layer sensor strip depicted in fig. 8A-8C. The graph is built up: the current values obtained after a very short incubation period of 0.25 seconds according to the invention can be correlated exactly with the actual plasma glucose concentration of the whole blood sample (R²＝0.999)。

Fig. 8E is a calibration curve obtained by plotting the end point currents (p4, p5, p6) for excitations 4, 5, and 6 obtained from the sensor strip depicted in fig. 8A-8C with a medium thickness reagent layer. The graph is built up: current values obtained according to the present invention after a very short initial incubation period of 0.25 seconds and a number of duty cycles including 0.5 second excitation and 0.25 second relaxation can be accurately correlated to the actual plasma glucose concentration of the whole blood sample (R²＝0.99)。

Certain definitions are set forth below to provide a clearer and more consistent understanding of the specification and claims.

A "sample" is a composition that may contain an unknown amount of analyte. The sample may contain water, such as whole blood, urine, saliva, or derivatives such as extracts, dilutions, filtrates, or reconstituted precipitates.

An "incubation period" is the length of time that a sample reacts with a reagent before an excitation is applied (such as before the first excitation is applied), and/or the time between excitations if the input signal includes multiple excitations.

A "measurable species" is any electrochemically active species that can be oxidized or reduced at the electrode surface at an appropriate potential.

"oxidoreductases" are capable of promoting the oxidation or reduction of an analyte or biological substrate. See, for example, Oxford Dictionary of Biochemistry and Molecular Biology, reviewed Edition, a.d. smith eds, New York: oxford University Press (1997), pages 161, 476, 477, and 560.

While various embodiments of the invention have been described, it will be apparent to those of ordinary skill in the art that other embodiments and implementations are possible within the scope of the invention.

Claims (24)

1. A method for determining an analyte concentration in a sample, comprising:

introducing the sample into a sensor strip;

applying a signal to the sample over an incubation period of up to 8 seconds;

generating a signal responsive to the concentration of the analyte in the sample and having a transient decay in response to a redox reaction of a measurable species, wherein a portion of the analyte in the sample generates the measurable species by the redox reaction; the transient decay is not inversely related to the square root of time and has a decay constant greater than or less than-0.5; and

the analyte concentration is determined from the transient decay of the generated signal by a correlation equation relating the generated signal to the analyte concentration in the sample.

2. The method of claim 1, wherein the measurable species comprises at least one mediator.

3. The method of claim 1, wherein the incubation period is at most 6 seconds.

4. The method of claim 1, wherein the incubation period is at most 4.75 seconds.

5. The method of claim 1, wherein the incubation period is 0.5 to 4 seconds.

6. The method of claim 1, wherein the transient decay is obtained within 0.5 seconds to 5 seconds of applying the signal to the sample.

7. The method of claim 1, wherein the transient decay is decreasing.

8. The method of claim 7, wherein the decreasing current decay is obtained within 0.5 seconds to 3 seconds of applying the signal to the sample.

9. The method of claim 7, wherein the analyte concentration is determined from a decreasing signal generated by a single excitation.

10. The method of claim 1, further comprising generating a changing concentration profile of the measurable species in a sample reservoir.

11. The method of claim 1, wherein the incubation period and application of the signal are completed in at most 6 seconds.

12. The method of claim 1, wherein the incubation period and application of the signal are completed in at most 4 seconds.

13. The method of claim 1, wherein the transient decay has a decay constant of-0.52 to-1.

14. The method of claim 1, wherein the transient decay has a decay constant of at most-0.35.

15. The method of claim 1, wherein the generated signal from which the analyte concentration is determined comprises a current value generated within 2 seconds of applying the signal to the sample.

16. The method of claim 1, wherein determining the analyte concentration from the generated signal is accomplished within 3 seconds of applying the signal.

17. The method of claim 1, wherein the sample is present in a reservoir defined by the sensor strip base and the bottom surface of the cover, wherein

The base is 50 to 150 microns from the bottom surface of the lid,

the volume of the sample within the reservoir is at most 3.5 microliters, and

the incubation period is at most 6 seconds.

18. The method of claim 17, wherein a reservoir height from the sensor strip base to the bottom surface of the lid is at most 100 microns, a volume of the sample within the reservoir is at most 3 microliters, the reservoir includes at least one reagent layer having an average initial thickness of at most 2 microns, and the incubation period is at most 2 seconds.

19. A method for determining an analyte concentration in a sample, comprising:

applying a signal to the sample during an incubation period of 0.4 to 1 second, the signal comprising at least three excitations, each excitation having a duration of 0.1 to 5 seconds;

generating a signal responsive to the concentration of the analyte in the sample and having a transient decay in response to the redox reaction of the measurable species, wherein a portion of the analyte in the sample generates the measurable species through the redox reaction, the transient decay not being inversely related to the square root of time and having a decay constant greater than or less than-0.5; and

the analyte concentration is determined from the transient decay of the generated signal by a correlation equation relating the generated signal to the analyte concentration in the sample.

20. The method of claim 19, wherein the excitations are separated by at least two relaxations, each relaxation having a duration of 0.1 to 3 seconds.

21. The method of claim 19, wherein the applied signal comprises at least 2 duty cycles applied within 5 seconds.

22. The method of claim 19, wherein signal application is completed in at most 4 seconds.

23. The method of claim 19, wherein the transient decay has a decay constant of-0.52 to-1.

24. The method of claim 19, wherein the transient decay has a decay constant of at most-0.35.