JP7371219B2 - Determination of contamination of biosensors used in analyte measurement systems - Google Patents

Description

TECHNICAL FIELD This application relates generally to analyte measurement systems, and more specifically to methods for determining contamination, such as moisture contamination of biosensors used in analyte measurement systems.

Detection of analytes in physiological fluids, such as blood or blood-derived products, is becoming increasingly important to today's society. Analyte detection assays are utilized in a variety of applications, including clinical laboratory testing, home testing, etc., and the results of such tests are important in the routine diagnosis and management of various disease states. plays a role. Analytical items of interest include glucose for diabetes management and cholesterol, among others. In response to the increasing importance of analyte detection, a variety of testing protocols and devices have been developed for both clinical and home use.

One of the methods employed for analyte detection in liquid samples is electrochemical methods. In such methods, an aqueous liquid sample, such as a blood sample, is deposited onto a biosensor and fills a sample receiving chamber of an electrochemical cell that includes two electrodes, eg, a counter electrode and a working electrode. The analyte is reacted with a redox reagent to form an oxidizing (or reducing) substance in an amount dependent on the analyte concentration. The amount of oxidizing (or reducing) material present is then estimated electrochemically and related to the amount of analyte present in the precipitated sample.

For example, LifeScan Inc. One of the blood glucose measurement systems manufactured by the company, sold as One-Touch Verio (hereinafter referred to as "Verio"), has shown excellent overall performance and accuracy.

However, any analyte measurement system may be susceptible to various modes of inefficiency and/or error. For example, biosensors used in analyte measurement systems, such as disposable test strips, can become contaminated or damaged when stored for patient-administered blood tests, such as blood glucose tests. Unfortunately, contaminated or damaged test strips can result in erroneous or higher than expected analyte concentration measurements. Such erroneous measurements can mislead the subject to administer the wrong amount of drug, with potentially devastating consequences. Therefore, there is an urgent need to determine whether a critical amount of contamination or damage has actually occurred to the biosensor before reporting analyte measurement results.

In order that the features of the invention may be understood, a detailed description can be obtained by reference to specific embodiments, some of which are illustrated in the accompanying drawings. The drawings, however, are only illustrative of particular embodiments and therefore should not be considered as limiting the scope of the disclosed subject matter, as it also encompasses other embodiments. Please note that. The drawings are not necessarily to scale and depict selected embodiments and are not intended to limit the scope of the invention. In the drawings, like numerals are used to indicate like parts throughout the various figures.

1 is a perspective view of an analyte measurement system including a test meter and a biosensor (test strip) in accordance with aspects described herein; FIG. 2 is a top view of a circuit board located in the test meter of FIG. 1 illustrating various components in accordance with aspects described herein; FIG. 3 is a perspective view of an assembled test strip suitable for use with the analyte measurement system of FIGS. 1 and 2; FIG. FIG. 3B is an exploded perspective view of the test specimen of FIG. 3A. FIG. 3B is an enlarged perspective view of the proximal portion of the test strip of FIGS. 3A and 3B. FIG. 3A is a bottom view of the test piece of FIGS. 3A-3C. 3A-3D are side elevational views of the specimens of FIGS. 3A-3D; FIG. FIG. 3A is a top view of the test piece of FIGS. 3A-3E. 3A-3F is a partial side elevational view of the proximal portion of the specimen of FIGS. 3A-3F; FIG. FIG. 3 is a simplified schematic diagram illustrating a test meter electrically interfacing with a portion of a test strip, such as the test strip shown in FIGS. 3A-3F. 5 shows an example of a test waveform applied by the test meter of FIG. 4 to the working and counter electrodes of a test strip at predetermined time intervals to determine an analyte in a sample applied to the test strip. Figure 5A shows the current measured over time based on the waveform of Figure 5A for a nominal specimen. 1 is a flowchart representing a method for determining analyte concentration in a test strip. 5A is a graphical comparison showing measured current values between a nominal test specimen and a contaminated test specimen over time, based on a portion of the waveform of FIG. 5A; FIG. 1 is a flowchart depicting a method for determining the presence of contamination in a test strip, according to embodiments described herein.

The following detailed description should be read with reference to the drawings, in which similar elements in different drawings are numbered the same. The drawings are not necessarily to scale and depict selected embodiments and are not intended to limit the scope of the invention. The detailed description serves to illustrate the principles of the invention by way of example and not by way of limitation. This description clearly enables those skilled in the art to make and use the invention, and describes several embodiments, applications, variations, alternatives, and uses of the invention, some of which include: It also includes what is currently considered to be the best mode for carrying out the invention.

As used herein, the term "about" or "approximately" in reference to any numerical value or range thereof means that a portion or collection of the components functions in accordance with the intended purpose set forth herein. Indicate appropriate dimensional tolerances to allow for Additionally, as used herein, the terms "patient," "host," "user," and "subject" refer to any human or animal subject for which the system or method is used in humans. Although not intended to be limiting, use of the subject technology in human patients represents a preferred embodiment.

The present disclosure relates, in part, to using a biosensor, such as a disposable test strip, to determine whether the biosensor is contaminated or damaged prior to conducting a test to determine the analyte concentration of an applied sample. Regarding technology for These techniques can be applied to specimens exposed to extreme temperatures (e.g., well above normal room temperature), excessive light, high levels of humidity, etc., in addition to moisture contamination. Such contamination or exposure, which may result from improper storage, can lead to the conversion of certain amounts of mediator onto the test strip electrode, for example from potassium ferrocyanide to potassium ferrocyanide. In one example, a blood glucose test strip contaminated with moisture may lead to a falsely higher result than expected, about 80 mg/dL (or more), which is higher than the actual blood glucose level. In such cases, this higher than expected reading could lead to administering an incorrectly high dose of insulin to the patient, which could seriously impact the patient's health.

Conversely, if small amounts of moisture can contaminate the test strip and the test strip still gives results within an acceptable precision range, the test results should be displayed to the patient. Therefore, simply determining that an unknown level of moisture has contaminated a test strip does not solve the problem of simply eliminating higher-than-expected results, increases the cost of blood glucose testing, and Outcomes are reduced. Additionally, techniques that require new test instruments or additional physical test specimens are not compatible with previously deployed units and also increase cost. Additionally, testing a test strip to determine contamination is only valid if the test itself does not damage or interfere with the use of the test strip to perform analyte measurements.

Although the Verio system described above has very good overall performance, however, tests have shown that the biosensor is not immune to contamination, such as contamination that can occur as a result of improper storage of test strips. It is not impermeable to the surface. Such contamination may include moisture contamination or contamination from other external sources or stimuli (temperature, light, humidity). In an attempt to find ways to reduce the effects of contamination, techniques are provided herein to alert users of test strips that produce erroneous results due to contamination based on storage and environmental conditions. Accordingly, various aspects of methods for determining whether a biosensor is contaminated are presented herein. In one example of this technology, the measurement of the analyte can be performed simultaneously with the determination of contamination, so that if the biosensor is not considered contaminated or damaged, the test results can be released (displayed) to the patient. Can be done. Also, if the test strip is deemed contaminated, test results can be suppressed to avoid giving the patient a higher than expected analyte reading that could lead to inappropriate dosing. Can be done.

Generally stated and according to at least one embodiment, a method for determining contamination of a biosensor is provided. The biosensor is loaded into the test meter and a sample is applied to the biosensor. applying a first predetermined voltage between spaced apart electrodes of the electrochemical cell for a first predetermined time interval; and applying a second predetermined voltage between the spaced apart electrodes during a second predetermined time interval after the first predetermined time interval. Apply voltage. A first current value is measured during a first predetermined time interval. The first reference value is determined based on the sum of the first current values during the first predetermined time interval. A second current value is measured during a second predetermined time interval. A second reference value is determined based on a peak current value measured during a second predetermined time interval. The third reference value is determined based on the rate of change of the current value measured after the peak current value during the second time interval. Determining whether the biosensor is contaminated can be determined based on one or more of the first to third reference values. Analyte concentration reporting is suppressed if the biosensor is determined to be contaminated. In another embodiment, a test meter is presented that performs the steps of the method described above.

The embodiments described above are intended to be illustrative only. It will be readily apparent from the discussion below that other embodiments are within the scope of the disclosed subject matter.

Next, specific examples of work will be explained with reference to FIGS. 1 to 6.

FIG. 1 shows a diabetes management system that includes a portable test meter 10 and a biosensor provided in the form of a disposable test strip 62 configured for the detection of blood sugar. For purposes of the following discussion, portable test meters will be referred to interchangeably throughout as analyte measurement management units, glucose meters, meters, and/or meter units. Although not shown in this figure, in at least one embodiment, the portable test meter may be combined with an insulin delivery device, an additional analyte testing device, and a drug delivery device. The portable test meter may be connected to a remote computer or server via a cable or via a suitable wireless technology such as GSM, CDMA, Bluetooth, WiFi, etc. . Such analyte measurement systems are disclosed in U.S. Pat. WO 2012/012341 entitled ``System and Method for Measuring an Analyte in the Sample'', each of which is incorporated herein by reference in its entirety.

Continuing to refer to FIG. 1, portable test meter 10 is defined by a housing 11 having a plurality of user interface buttons (16, 18, 20) disposed on facing surfaces. Display 14 is provided in addition to a test strip port opening 22 configured to receive a biosensor (test strip 62). User interface buttons (16, 18, 20) may be configured to allow data entry, menu navigation, and command execution. It will be readily appreciated that the configuration and functionality of the user interface buttons of portable test meter 10 are intended as examples and that modifications and variations are possible. According to this particular embodiment, user interface button 18 may be in the form of a two-way toggle switch. The data may include values representative of analyte concentrations and/or information related to the individual's daily life. Information regarding daily life may include an individual's dietary intake, medication status, health checkup status, health status, and exercise level.

As shown in FIG. 2 and shown in simplified schematic form, the electronic components of the portable test meter 10 are located on the top surface of a circuit board 34 contained within the housing 11 of FIG. has been done. In this embodiment, these electronic components include test strip port connector 23, operational amplifier circuit 35, microcontroller 38, display connector 14a, non-volatile memory 40, clock 42, and first wireless module 46. On the opposing bottom surface of circuit board 34, electronic components may include a battery connector (not shown) and data port 13. It will be appreciated that the relative positions of the various electronic components may vary, and the configurations described herein are exemplary.

The microcontroller 38 includes a specimen port connector 22 aligned with the specimen port opening 23 (FIG. 1), an operational amplifier circuit 35, a first wireless module 46, a display 14, a non-volatile memory 40, a clock 42, and at least one It may be electrically connected to two batteries (not shown), data port 13, and user interface buttons (16, 18, and 20).

Op amp circuit 35 may include two or more op amps configured to provide some of the potentiostat functionality and current measurement functionality. Potentiostatic function may refer to the application of a test voltage between at least two electrodes of a test strip. Current function may refer to the measurement of test current resulting from an applied test voltage. Current measurements may be performed using a current to voltage converter. Microcontroller 38 may be in the form of a mixed signal microprocessor (MSP), such as the Texas Instrument (TI) MSP430, for example. MSP 430 may also be configured to perform some potentiostat functions and current measurement functions. Further, 430 may include volatile memory and non-volatile memory. In another embodiment, many of the electronic components may be integrated with the microcontroller in the form of an application specific integrated circuit (ASIC).

Test strip port connector 22 may be configured to form an electrical connection to test strip 62. Display connector 14a may be configured to attach to display 14. For purposes of this description, display 14 is in the form of a liquid crystal display for reporting measured glucose levels and for facilitating input of lifestyle-related information using interface buttons 16, 18, 20. It may be. Display 14 may optionally include a backlight. Data port 13 can accept a suitable connector attached to a connecting lead, thereby allowing test meter 10 to be linked to an external device such as a personal computer (not shown). For purposes of this description, data port 13 may be any port that allows the transmission of data, such as, for example, a serial, USB, or parallel port. Data port 13 is accessible through housing 11 of portable test meter 10. Clock 42 may be configured to keep a current time and measure time relative to the geographic region in which the user is located, as described in more detail below. The test meter may be configured to be electrically connected to a power source, such as, for example, at least one included battery (not shown).

3A-3G show various views of test strips 62 suitable for use with the methods and systems described herein. In the exemplary embodiment, test strip 62 is defined by an elongated body extending from a distal end 80 to an opposing proximal end 82 and having lateral edges 56, 58, as shown in FIG. 3A. As shown in FIG. 3B, the test strip 62 is also sandwiched between a first electrode layer 66, a second electrode layer 64, and two electrode layers 64, 66 at a distal end 80 of the test strip 62. It includes a spacer 60. The first electrode layer 66 may include a first electrode 66, a first connection track 76, and a first contact pad 67, the first connection track 76 being as shown in FIGS. 3B and 3C. , electrically connecting the first electrode 66 with the first contact pad 67 . Note that the first electrode 66 is a portion of the first electrode layer 66 directly under the reagent layer 72, as shown in FIGS. 3A and 3B. Similarly, the second electrode layer 64 may include a second electrode 64, a second connection track 78, and a second contact pad 63, with the second connection track 78 shown in FIGS. 3A-3C. As shown, the second electrode 64 is electrically connected to the second contact pad 63. Note that the second electrode 64 is a portion of the second electrode layer 64 disposed above the reagent layer 72, as best shown in FIGS. 3B and 3C.

As shown in FIGS. 3B-3E, a sample receiving chamber 61 (e.g., an electrochemical cell) is defined by a first electrode 66, a second electrode 64, and a spacer 60 proximate a distal end 80 of a test strip 62. be done. First electrode 66 and second electrode 64 may define the bottom and top, respectively, of sample receiving chamber 61, as shown in FIG. 3G. Cutout region 68 of spacer 60 may define a sidewall of sample receiving chamber 61, as shown in FIG. 3G. In one aspect, sample receiving chamber 61 may include a port 70 that provides a sample inlet and/or vent, as shown in FIGS. 3A-3C. For example, one of the ports 70 may allow entry of a fluid sample and the other port 70 may allow exit of air.

In exemplary embodiments, sample receiving chamber 61 may have a small volume. For example, chamber 61 may have a volume ranging from about 0.1 microliters to about 5 microliters, about 0.2 microliters to about 3 microliters, or preferably about 0.3 microliters to about 1 microliter. May have. To provide a small sample volume, the cutout 68 is about 0.01 cm ² to about 0.2 cm 2 , about 0.02 ^{cm 2 to} about 0.15 cm ² , or preferably about 0.03 cm ² to about It may have an area in the range of 0.08 ^cm2 . Further, the first electrode 66 and the second electrode 64 are in the range of about 1 micrometer to about 500 micrometers, preferably in the range of about 10 micrometers to about 400 micrometers, more preferably in the range of about 40 micrometers to about They may be spaced in the range of 200 micrometers. Also, the relatively close spacing of the electrodes may allow a redox cycle to occur, in which the mediator produced and oxidized at the first electrode 66 diffuses to the second electrode 64 and is reduced. , which can then diffuse into the first electrode 66 and be oxidized again. Those skilled in the art will appreciate that a variety of such volumes, areas, and/or spacings of electrodes are within the spirit and scope of the present disclosure.

In one embodiment, first electrode 66 and second electrode 64 may each include an electrode layer. The electrode layer may include a conductive material formed from materials such as gold, palladium, carbon, silver, platinum, tin oxide, iridium, indium, or combinations thereof (eg, indium-doped tin oxide). Additionally, the electrode layer may be formed by placing a conductive material on an insulating sheet (not shown) by sputtering, electroless plating, or screen printing processes. In one exemplary embodiment, first electrode 66 and second electrode 64 may include electrode layers made from sputtered palladium and sputtered gold, respectively. Suitable materials that may be employed as spacer 60 include various insulating materials such as, for example, plastics (eg, PET, PETG, polyimide, polycarbonate, polystyrene), silicone, ceramics, glass, adhesives, and combinations thereof.

In one embodiment, spacer 60 may be in the form of a double-sided adhesive coated on opposite sides of a polyester sheet, and the adhesive may be pressure sensitive or heat activated. Applicants note that various other materials for first electrode layer 66, second electrode layer 64, and/or spacer 60 are within the spirit and scope of this disclosure.

Either the first electrode 66 or the second electrode 64 can serve as a working electrode depending on the magnitude and/or polarity of the at least one applied test voltage. The working electrode may measure a limit test current that is proportional to the reduced mediator concentration. For example, if the current-limiting species is a reduced mediator (e.g., potassium ferrocyanide), as long as the test voltage is sufficiently greater than the redox mediator potential with respect to the second electrode 64, it will not be oxidized at the first electrode 66. It's okay. In this situation, the first electrode 66 acts as the working electrode and the second electrode 64 acts as the counter/reference electrode. Applicants note that the counter/reference electrode may also be referred to simply as the reference electrode or the counter electrode. Critical oxidation occurs when all of the reduced mediator is depleted at the surface of the working electrode, such that the measured oxidation current is proportional to the flux of reduced mediator diffusing from the bulk solution towards the working electrode surface. Become. As used herein, the term "bulk solution" refers to the portion of the solution sufficiently distant from the working electrode that the reduced mediator is not located within the depleted region. Note that unless otherwise noted for test strip 62, all potentials applied by test meter 10 are described below with respect to second electrode 64.

Similarly, if the test voltage is sufficiently smaller than the redox mediator potential, reduced mediator may be oxidized at the second electrode 64 as a limiting current. In such a situation, second electrode 64 acts as a working electrode and first electrode 66 acts as a counter/reference electrode.

Initially, analysis may involve introducing a volume of fluid sample into sample receiving chamber 61 through one of ports 70. In one aspect, port 70 and/or sample receiving chamber 61 may be configured such that a fluid sample fills sample receiving chamber 61 by capillary action. The first electrode 66 and/or the second electrode 64 may be coated with a hydrophilic reagent to promote capillary action in the sample receiving chamber 61. For example, a thiol derivatizing reagent having a hydrophilic moiety, such as 2-mercaptoethanesulfonic acid, may be coated on the first electrode and/or the second electrode.

In the analysis of the test strip 62, the reagent layer 72 may include glucose dehydrogenase (GDH) based on PQQ coenzyme and ferricyanide. In another embodiment, the PQQ coenzyme-based enzyme GDH can be replaced with the FAD coenzyme-based enzyme GDH. When blood or control solution is dispensed into the sample reaction chamber 61, glucose is oxidized by GDH _(ox) and in the process the following chemical changes T. 1, GDH _(ox) is converted to GDH _(red) . Note that GDH _(ox) means the oxidized state of GDH, and GDH _(red) means the reduced state of GDH.

T. 1 D-glucose + GDH _(ox) → gluconic acid + GDH _(red)

Next, GDH _(red) undergoes the following chemical change T. 2, it is regenerated to the active oxidized state by ferricyanide (ie, oxidized mediator or Fe(CN) ₆ ^3- , such as potassium ferricyanide). In the process of regenerating GDH _(ox) , T. 2 produces ferrocyanide (ie, reduced mediator or Fe(CN) ₆ ^4- , eg, potassium ferrocyanide).

T. 2 GDH _(red) +2Fe(CN) ₆ ^3- →GDH _(ox) +2Fe(CN) ₆ ^4-

FIG. 4 provides a simplified schematic diagram showing test meter 10 in conjunction with first contact pads 67a, 67b and second contact pads 63 of test strip 62. FIG. The second contact pad 63 may be used to establish an electrical connection with the test meter 10 via the U-shaped notch 65, as illustrated in FIG. 3B. In one embodiment, the test meter 10 includes a second electrode connector 101, a first electrode connector (102a, 102b), a test voltage unit 106, a current measurement unit 107, a processor, as shown schematically in FIG. 212, a memory unit 210, and a visual display 202. First contact pad 67 may include two prongs labeled 67a and 67b. In one exemplary embodiment, first electrode connectors 102a and 102b connect to prongs 67a and 67b, respectively, separately. The second electrode connector 101 may connect to the second contact pad 63. Test meter 10 may measure resistance or electrical continuity between prongs 67a and 67b to determine whether test strip 62 is electrically connected to test meter 10.

In one embodiment, test meter 10 may apply a test voltage and/or current between first contact pad 67 and second contact pad 63. When test meter 10 recognizes that test strip 62 has been inserted, test meter 10 turns on and enters a fluid detection mode. In one embodiment, the fluid detection mode causes test meter 10 to apply a constant current of about 1 microamp between first electrode 66 and second electrode 64. Since test strip 62 is initially dry, test meter 10 measures a relatively large voltage. When the fluid sample bridges the gap between the first electrode 66 and the second electrode 64 during the dosing process, the test meter 10 detects a temperature below a predetermined threshold that causes the test meter 10 to automatically initiate a glucose test. We will measure the decrease in the measured voltage that is .

A method for determining analyte concentration using test strip 62 and test meter 10 is described below with reference to FIGS. 5A-5C. As an overview, first, application of test voltage and measurement of current value will be explained, and then analyte concentration measurement will be explained.

First, regarding the application of voltage to the test piece, reference will be made to the test measuring instrument 10 of the example and the test piece 62 of the example. Test meter 10 may include electronic circuitry that can be used to apply a plurality of voltages to test strip 62 and measure current transient output resulting from an electrochemical reaction within the test chamber of test strip 62. . Test meter 10 may also include a signal processor having a set of instructions for a method of determining analyte concentration in a fluid sample as disclosed herein. In one embodiment, the analyte is blood sugar.

Continuing the discussion of applying test voltages, FIG. 5A shows an exemplary waveform of multiple test voltages applied to a test strip 62 at predetermined time intervals. The plurality of test voltages according to this waveform include a first test voltage E1 applied over a first time interval _t1 , a second test voltage E2 applied over a second time interval _t2 , and a third test voltage E2 applied over a second time interval t2. includes a third test voltage E3 applied over an interval _t3 . The third voltage E3 may differ from the second test voltage E2 in the magnitude of the electromotive force, the polarity, or a combination of both. As in the preferred embodiment and illustrated, E3 may be the same size as E2, but with opposite polarity. The glucose test time interval t _G represents the amount of time to perform a glucose test (but not necessarily to perform all calculations associated with a glucose test). The glucose test time interval t _G may range from about 1.1 seconds to about 5 seconds. Further, as shown in FIG. 5A, the second test voltage E2 may include a constant (DC) test voltage component and a superimposed alternating (AC) or alternating oscillating test voltage component applied at short time intervals. . More specifically, superimposed alternating or oscillating test voltage components may be applied over a time interval indicated by _tcap at the beginning of a second time interval.

The plurality of test current values measured during any time interval is at a frequency within the range of about 1 measurement per microsecond to about 1 measurement per 100 milliseconds, preferably about 10 milliseconds. It may be executed with Although embodiments are described that use three test voltages in sequence, glucose tests may include different numbers of open circuits and test voltages. For example, in an alternative embodiment, a glucose test may include an open circuit during a first time interval, a second test voltage during a second time interval, and a third test voltage during a third time interval. Note that references to "first," "second," and "third" are chosen for convenience and do not necessarily reflect the order in which the test voltages are applied. For example, one embodiment may have a potential waveform in which the third test voltage may be applied before the first and second test voltages are applied.

FIG. 5C is a flowchart depicting a method 500 for determining analyte concentration in a test strip based on the waveform of FIG. 5A and the measured current as shown in FIG. 5B. At exemplary step 510, a glucose assay begins by inserting a test strip 62 into test meter 10 and depositing a sample onto test strip 62. At exemplary step 520, test meter 10 conducts a first test between first electrode 66 and second electrode 64 for a first time interval t ₁ (e.g., 1 second in FIG. 5A). A voltage E1 (eg, approximately 20 mV in FIG. 5A) may be applied. The first time interval t ₁ may range from about 0.1 seconds to about 3 seconds, preferably from about 0.2 seconds to about 2 seconds, and most preferably from about 0.3 seconds to about 2 seconds. The range is approximately 1.1 seconds.

The first time interval t ₁ may be long enough so that the sample receiving chamber 61 is completely filled with sample and the reagent layer 72 is at least partially dissolved or solvated. In one aspect, the first test voltage E1 may be relatively close to the redox potential of the mediator such that a relatively small amount of reduction or oxidation current is measured. FIG. 5B shows that a relatively small amount of current is observed during the first time interval t1 compared to the second time interval _t2 and the _third time interval _t3 . For example, when using potassium ferricyanide and/or potassium ferrocyanide as the mediator, the first test voltage E1 in FIG. 5A ranges from about 1 mV to about 100 mV, preferably from about 5 mV to about 50 mV, and most preferably from about It may range from 10 mV to about 30 mV. Although in the preferred embodiment the applied voltage is given as a positive value, the same voltage in the negative region may also be utilized to achieve the intended purpose of the claimed invention. During this time interval, the first current output may be sampled by the processor at step 530 to collect current values over the time interval.

In exemplary step 540, after applying the first test voltage E1 (step 520) and sampling the output (step 530), test meter 10 applies the first test _voltage E1 (step 520) and samples the output (step 530). A second test voltage E2 (eg, about 3 millivolts in FIG. 5A) is applied between the first electrode 66 and the second electrode 64 for about 3 seconds). The second test voltage E2 may be of a different value than the first test voltage E1 and is sufficiently negative with respect to the mediator redox potential such that the limiting oxidation current is measured at the second electrode 64. It's good. For example, when using potassium ferricyanide and/or potassium ferrocyanide as the mediator, the second test voltage E2 may range from about 0 mV to about 600 mV, preferably from about 100 mV to about 600 mV, and more preferably It is about 300mV.

The second time interval t ₂ is preferably long enough so that the rate of production of reduced mediator (eg, potassium ferrocyanide) can be monitored based on the magnitude of the critical oxidation current. The reduced mediator is produced by an enzymatic reaction with the reagent layer 72. During a second time interval _t2 , a critical amount of reduced mediator is oxidized at the second electrode 64, a non-critical amount of oxidized mediator is reduced at the first electrode 66, and a critical amount of the oxidized mediator is reduced at the first electrode 66. A concentration gradient is formed between electrode 66 and second electrode 64.

In an exemplary embodiment, the second time interval t ₂ is also such that a sufficient amount of potassium ferricyanide can be diffused to the second electrode 64 or from the reagent on the first electrode. It is preferable that the length be sufficiently long. A sufficient amount of potassium ferricyanide is required at the second electrode 64 such that a limiting current for oxidizing the potassium ferricyanide at the first electrode 66 can be measured during the third test voltage E3. This is for the purpose of The second time interval t ₂ may be less than about 60 seconds, preferably in the range of about 1.1 seconds to about 10 seconds, more preferably in the range of about 2 seconds to about 5 seconds. Similarly, the time interval shown as t _cap in FIG. 5A may also last for a range of time, but in one exemplary embodiment, its duration is approximately 20 milliseconds. In one exemplary embodiment, the superimposed alternating test voltage components are applied from about 0.3 seconds to about 0.4 seconds after the application of the second test voltage E2 and at about 109 Hz with an amplitude of about ±50 mV. Induces a sine wave with a frequency. During this time interval, the second current output may be sampled by the processor at step 550 to collect current values over the time interval.

FIG. 5B shows that the current i _pb is relatively small after the start of the second time interval t ₂ and then during the second time interval t ₂ the absolute value of the oxidation current gradually increases. A small peak i _pb occurs due to the oxidation of the endogenous or exogenous reducing agent after the transition from the first voltage E1 to the second voltage E2, and during the second time interval t ₂ the absolute value of the oxidation current resulting in a gradual increase in A small peak i _pb occurs after the change from the first voltage E1 to the second voltage E2, referred to herein as the voltage change line TL, due to the initial depletion of the reduced mediator. Thereafter, a slow absolute decrease in the oxidation current occurs after a small peak i _pb caused by the production of potassium ferrocyanide by the reagent layer 72 , followed by potassium ferrocyanide diffusion into the second electrode 64 .

In exemplary step 560, after applying the second test voltage E2 (step 540) and sampling the output (step 550), the test meter 10 connects the first electrode 66 and the second electrode 64. A third test voltage E3 (eg, approximately −300 millivolts in FIG. 5A) is applied during a third time interval t ₃ (eg, 1 second in FIG. 5A). The third test voltage E3 may be a sufficiently positive value of the mediator redox potential such that a critical oxidation current is measured at the first electrode 66. For example, when using potassium ferricyanide and/or potassium ferrocyanide as the mediator, the third test voltage E3 may range from about 0 mV to about -600 mV, preferably from about -100 mV to about -600 mV; More preferably about -300 mV.

After applying the third test voltage E3, in step 570, the current value is measured at a third time interval _t3 . The third time interval _t3 may be long enough to monitor the diffusion of the reduced mediator (eg, potassium ferrocyanide) near the first electrode 66 based on the magnitude of the oxidation current. During a third time interval _t3 , a critical amount of reduced mediator is oxidized at the first electrode 66 and a non-critical amount of oxidized mediator is reduced at the second electrode 64. The third time interval _t3 ranges from about 0.1 seconds to about 5 seconds, preferably from about 0.3 seconds to about 3 seconds, more preferably from about 0.5 seconds to about 2 seconds. It may be a range.

FIG. 5B shows that for the nominal specimen, the peak i _pc is relatively large at the beginning of the third time interval t ₃ and then decreases to a near steady-state current i _ss value. In one embodiment, the second test voltage E2 may have a first polarity and the third test voltage E3 may have a second polarity opposite to the first polarity. In another embodiment, the second test voltage E2 may be sufficiently negative of the mediator redox potential and the third test voltage E3 may be sufficiently positive of the mediator redox potential. The third test voltage E3 may be applied immediately after the second test voltage E2. However, those skilled in the art will appreciate that the magnitude and polarity of the second and third test voltages can be selected depending on the method by which the analyte concentration is determined.

Glucose concentration determination will now be described for embodiments described herein, as defined in step 580 of FIG. 5C. 5A and 5B show the sequence of events during the specimen transition period. Approximately 1.1 seconds after the start of the test sequence (and just after applying the second voltage E2 to make the second electrode the working electrode), the reagent has not yet reached the first electrode and the current is probably Current measurements are taken to later correct for interferences due to interfering reducing agents in the plasma (in the absence of mediator). Between about 1.4 seconds and about 4 seconds, when the mediator and oxidized mediator are able to diffuse into the second electrode (at least during the latter part of this time interval when the second voltage E2 is applied). At , a first glucose proportional current i _l is measured. Immediately after making the first electrode the working electrode by application of the third voltage E3, two single point measurements (at about 4.1 seconds and about 5 seconds according to the present embodiment) and one integrated measurement i _r will be held. Measurements sampled at 1.1 s, 4.1 s, and 5 s, respectively, according to this specific embodiment to correct i _r for the additional current (i _2corr ) from the interfering reductant. used for. The ratio of i _l and i _r is used to correct i _2corr for hematocrit interference effects.

が、グルコース濃度を決定するために使用される。式中、Ｇ_{ｂａｓｉｃ}は、分析物濃度で、
ｉ_ｒは、第三の時間間隔の間の第三の電流値の合計で、
ｉ_ｌは、第二の時間間隔における第二の電流値の合計で、

で、
ａ、ｂ、ｐ及びｚ_ｇｒは、所定の係数である。 In one embodiment, then the following equation

is used to determine glucose concentration. where G _basic is the analyte concentration;
i _r is the sum of the third current values during the third time interval;
i _l is the sum of the second current values in the second time interval,

in,
a, b, p and z _gr are predetermined coefficients.

である。 In one specific example,

It is.

In another example, a different test strip chemistry may be used, in which case the time appearing for the current evaluation is varied according to the general relationship described above. Additional details related to applied waveforms and determining test strip analyte concentrations are provided in U.S. Patent No. 8,709,232 and WO 2012/012341, previously incorporated herein by reference. has been done.

As mentioned above, FIG. 6A details an enlarged partial view of the current versus time based on the waveform of FIG. 5A. In this figure, the current response of FIG. 5B is reproduced for a nominal (uncontaminated) specimen (such as specimen 62 of FIG. 1) compared to the current response of a moisture-contaminated specimen. As clearly shown in this figure, there are many characteristic anomalies between the nominal specimen and the abnormal/defective specimen over a portion of the current transient. More specifically, the contaminated test strip includes a plurality of spike current transients exhibiting between about 0 seconds and 1 second (during the predetermined first time interval). Furthermore, the contaminated specimen exhibits a peak value i _pb that decreases after application of the test voltage at the beginning of the second time interval, approximately 1 second after the start of the test sequence.

Without being limited to any particular theory, the physical mechanism of moisture contamination is that the introduction of moisture (due to storage conditions or other sources) inhibits the conversion of potassium ferricyanide to potassium ferrocyanide in the reagent layer of the specimen. This seems to be what causes it. In such a case, the reagent layer will have a high concentration of potassium ferrocyanide that can be consumed by diffusion at both the first electrode surface and the second electrode surface during analyte concentration measurements. Therefore, the analyte signal will be amplified, resulting in a higher than expected glucose reading when the test strip becomes contaminated.

As verified by the experiments described below, there are many discrete and distinguishable anomalies in the first and second time intervals of the test waveform due to contamination (moisture) effects. These effects are illustrated for comparison in FIG. 6A. This contamination may be characterized by both physical and chemical changes to the specimen. For example, a physical change can effectively cause a specimen that, before contamination or damage, contained an electrode coated with a uniform layer of mediator, to now effectively appear as a rough or mismatched layer of unconverted mediator. It occurs because there is. In such a case, when a blood sample is applied to the test strip, the misalignment of this layer of the test strip may result in a current transient as observed in the first time interval.

Additionally, the specimen may also undergo chemical changes. These chemical changes, due to the overall amount of mediator transformed, result in specific and discernible changes in the expected current response of the specimen to the applied voltage, and more specifically to the second test voltage. It has the potential to bring about change. Although the combination of both physical and chemical changes to a contaminated or damaged specimen has been described in general terms, techniques for determining contamination are not limited by any particular aspect of this discussion.

As a result of the perceptible difference between the expected current response of the nominal test strip and the current response exhibited by the contaminated test strip, a number of reference values, conveniently labeled AE according to FIG. For this purpose, it may be applied when the test strip is inserted into a portable test meter. According to one embodiment, it was determined that identification of certain aspects of the abnormal current response (depicted as reference values A, B, and C) may be sufficient to determine the presence of a contaminated specimen.

According to one embodiment, the reference value A is the sum of the current values measured during a first time interval, for example 0.20 to 0.75 seconds. As mentioned above, the contaminated specimen exhibits a physical change that results in a larger current response during the first time interval due to the mediator layer losing its physical integrity. The sum of the current values in the first time interval therefore indicates the magnitude of these physical changes to the specimen due to contamination and serves as the reference value A. In one embodiment, a contaminated test strip exhibits a sum of current values between 0.20 and 0.75 seconds of an amount greater than 6.5 mA.

As mentioned above, contaminated specimens exhibit a smaller peak current i _bc at 1.0 seconds due to chemical changes in the specimen caused by contamination. Such contamination results in peak current deviations. In one embodiment, if the measured peak current value i _pb during the second time interval is less than 12.5 μA, a chemical change consistent with contamination is indicated, and this peak value indicates the reference value B.

Furthermore, the chemical change also leads to a slower rate of negative change in the measured current following the peak current. Thus, if the peak is at 1.05 seconds, the difference in value between 1.10 seconds and 1.05 seconds is an indication of this rate of change, referred to herein as reference value C. Therefore, the specimen can be further characterized in that the reference value C is the difference between the current value at 1.10 seconds and the current value at 1.05 seconds. In one embodiment, this difference may be between 0 and -3.5 μA. In another example, this difference may be divided by the time difference between the two values (eg, 1.10 seconds - 1.05 seconds = 0.05 seconds) to give the rate of change of current over time. Referring to FIG. 6B, reference values B and C may be determined at step 660.

In one embodiment, reference values B and C may be used in conjunction with reference value A to determine contamination of a test strip, such as in step 670 of FIG. 6B. Upon determining that the test strip is contaminated, in step 670 of FIG. 6, the meter may display or announce a message indicating that the test strip is contaminated. Advantageously, determination of test strip contamination allows for education of test meter users. Information may be provided to the user regarding the proper storage of the test strip, including the need to store the test strip in the provided closed container and away from excessive heat or light. Educate your users.

In one embodiment, upon determining contamination of a particular test strip using the flags described above, the test measurement system may invalidate the test results from the contaminated biosensor and a new biosensor may be placed for testing. should be loaded. If the new biosensor does not exhibit waveform characteristics associated with contamination, the test and measurement system can then notify the patient of the test results. In another embodiment, automatic delivery of insulin may occur to the patient only if the biosensor was not contaminated as determined by the techniques described above.

In another embodiment, a further refinement utilizes the observation that the contaminated test specimen is characterized as having a wider range of current values during the first interval than the nominal test specimen. Specifically, the range is defined as the difference between the highest current value and the lowest current value of the transient current exhibited in the contaminated specimen during the first time interval. Therefore, the reference value D is defined as the difference between the maximum current value and the minimum current value in the first time interval. In one embodiment, for contaminated test strips, the range of this difference, ie, reference value D, is greater than 0.57 μA, and for nominal test strips, this range is less than 0.57 μA.

In an additional embodiment, another improvement eliminates false positive contamination determinations by checking whether a contaminated test strip exhibits a current that is consistent with movement of the test strip within the meter under test. For example, finger movements relative to the specimen under test may cause some current excursion during the testing process. Therefore, a reference value E may be defined in which the minimum value of the current in the first time interval is greater than 0 μA. Referring to FIG. 6B, reference values A, D, and E may be determined at step 640.

Given the definitions of reference values A to E, a set of flags (Flag _A to Flag _E ) can be used to distinguish between nominal specimens (FIGS. 5B and 6A) and abnormal specimens (FIG. 6A) for the purpose of the analyte measurement system. may be defined based on a perceivable representative difference between Each of the flags is a Boolean flag that can be either true or false, and each flag A-E sets a respective reference value A-E to a respective range defined by a respective target value A-E or It is based on comparing values.

Flag _A is TRUE if the reference value A, defined as the sum value over a portion of the first time interval, eg, between 0.20 and 0.75 seconds, is greater than the target value A. The target value A in this specific example is 6.5 μA. However, a target value A in the range of 5-10 μA was found to provide sufficient effectiveness.

Flag _B is TRUE if the reference value B defined as the measured peak current value i _pb during the second time interval is smaller than the target value B approximately. The target value B in this specific example is 12.5 μA. However, a target value B in the range of 12-12.5 μA was found to provide sufficient effectiveness for the purpose of identifying abnormal specimens.

Flag _C indicates that the reference value C, defined as the difference in current value between the measured peak current value i _pb (e.g. at 1.05 seconds) and the current value at 1.10 seconds, is 0 and the target value C. If it is between then it is TRUE. The target value C is −3.5 μA in this specific example. However, a target value C in the range of about 0 to -4.5 μA was found to provide sufficient effectiveness for the purpose of identifying contaminated specimens.

Flag _D is TRUE if the reference value D, which is defined as the difference between the maximum current value and the minimum current value in the first time interval, is greater than about the target value D. A target value D in the range of approximately 0.4-0.65 mA was found to provide adequate effectiveness. According to this specific example, the target value D is 0.57 μA.

Finally, Flag _E is TRUE if the reference value E, defined as the minimum transient current in the first time interval, is greater than the target value E, for example, greater than about 0 mA, as in this specific example. It is.

In one embodiment, a determination of contamination or damage to a test specimen may be made when one or more of Flag _A -Flag _E , eg, only Flag _A , Flag _B , and Flag _C evaluate to true. In another embodiment, a determination of test specimen contamination or damage may be made when all of Flags _A through Flag _E evaluate to true. For example, Flags A, D, and E can be considered to represent physical changes due to contamination as described above, and Flags B and C can be considered to represent chemical changes due to contamination as described above. In such cases, a combination of at least one flag from each group, i.e. one of Flags A, D and E and one of Flags B and C, is used to detect physical and chemical changes to the specimen. Contamination can be determined by a combination of

Of further note, Flags A, D and E occur earlier in the test sequence than Flags B and C, thus eliminating the influence of false positives due to blood filling issues from the finger or specimen movement/nudging during testing. It may be easier to receive. As an advantage, the present technique can combine the flags of each group to eliminate such false positives, and uncontaminated specimens are not wasted due to this false positive. Furthermore, the selection of target values AE in the ranges described above advantageously provides a balance between the desired result of capturing as many true positives as possible while avoiding as many false positives as possible.

Unexpectedly, various excursions in the output waveform occur during testing of specimens intentionally exposed to moisture, including the transient current values discussed above with respect to FIG. 6A. While there are some potential variations that can be experienced based on application of a sample from a fingertip as opposed to a pipette due to variations in filling rate, for example, changes in physical properties in the reagent layer as discussed above. A demonstrable change in current response from the nominal current response in Figure 5B due to the current response may also be observed.

To verify the reliability of the technique described above, tests were conducted on 92 contaminated specimens. The specimens were determined to be contaminated because they had been stored in a container containing a desiccant and the desiccant was tested and found to contain moisture. A test meter was used to apply a voltage to the specimen and its output current was captured as described herein. First, conventional techniques for detecting test errors were applied to the acquired current and a total of 39 of the contaminated specimens were identified as having errors related to other factors such as filling. When the technique was applied to the acquired transient currents using a combination of flags A, B, C, D, and E, all 92 contaminated specimens were properly identified based on the criteria described above. .

Although the invention has been described with respect to particular variations and exemplary drawings, those skilled in the art will recognize that the invention is not limited to the variations or drawings described. In addition, where the methods and steps described above indicate that certain events occur in a certain order, those skilled in the art will also recognize that the order of the certain steps may be changed and that such changes may be considered variations of the present invention. You will also recognize that this is by example. Additionally, certain of the steps may not only be performed in the order as described above, but also simultaneously in parallel processes where possible. Accordingly, this patent is intended to cover variations of the invention to the extent that there are variations of the invention that fall within the spirit of the disclosure or are equivalent to the invention found in the claims. intended.

To the extent that the claims recite the phrase "at least one of" with respect to multiple elements, this is intended to mean at least one or more of the listed elements, and at least one of each element. Not limited to one. For example, "at least one of element A, element B, and element C" is intended to refer to element A only, or element B only, or element C only, or any combination thereof. "At least one of element A, element B, and element C" is not intended to be limited to at least one of element A, at least one of element B, and at least one of element C.

This written description uses examples to disclose the invention, including the best mode, and also to explain how any person skilled in the art can make and use any apparatus or system or carry out any method employed. This will enable the present invention to be implemented. The patentable scope of the invention is defined by the claims, and may include other examples that occur to those skilled in the art, where such other examples have structural elements that do not differ from the literal language of the claims. , or equivalent structural elements that do not materially differ from the literal language of the claims, are intended to be included within the scope of the claims.

The terminology used herein is for the purpose of describing particular embodiments only and is not intended to be limiting. As used herein, the singular forms "a, an" and "the" are intended to include the plural forms as well, unless the context clearly dictates otherwise. "comprise" (and any form of "comprise" as in "comprises" and "comprising"), "have" (as in "has" and "having") ), "include" (and any form of include, such as "includes" and "including"), and "contain" (as well as "contains" and "containing"). Any form of the term contain is further understood to be an open-ended linking verb, such as "comprising," "having," or "having" one or more steps or elements. A method or apparatus that "comprises" or "contains" retains one or more steps or elements thereof, but is not limited to retaining one or more of those steps or elements. Similarly, one or more An element of a method step or apparatus that "comprises," "has," "includes," or "contains" retains one or more of those characteristics, but retains one or more of those characteristics. Further, a device or structure configured in a particular manner may be configured in at least that manner, but may also be configured in ways not listed.

Corresponding structures, materials, acts, and equivalents of all means-plus-function or step-plus-function elements of the following claims, if any, are covered by other specifically claimed claims, if any. Intended to include any structure, material, or act that, in combination with the elements, performs a function. The description herein is presented for purposes of illustration and description, and is not intended to be exhaustive or limited to the form disclosed. Many modifications and variations will be apparent to those skilled in the art without departing from the scope and spirit of this disclosure. The embodiments have been selected and described to best explain the principles and practical application of one or more aspects described herein, and that others skilled in the art will understand as suitable for the particular use contemplated. This is provided to provide an understanding of one or more aspects described herein with various modifications.

Claims (19)

A method for determining the presence of contamination of a biosensor, the method comprising:
loading the biosensor into a test meter and applying a sample to the biosensor, the biosensor having an electrochemical cell defined by a pair of spaced apart electrodes;
A first predetermined voltage is applied between the spaced apart electrodes of the electrochemical cell for a first predetermined time interval, and a second predetermined voltage is applied between the spaced apart electrodes a second predetermined time interval after the first predetermined time interval. applying the voltage over two predetermined time intervals;
sampling and measuring a first current value during the first predetermined time interval;
determining a first reference value based on a total value that is the sum of the first current values measured during the first predetermined time interval;
measuring a second current value during the second predetermined time interval;
a second reference value based on a peak current value measured during the second predetermined time interval and a rate of change of the current value measured after the peak current value during the second predetermined time interval; a step of determining a third reference value based on;
determining whether the biosensor is contaminated based on one or more of the first to third reference values;
suppressing reporting of analyte concentration when the biosensor is determined to be contaminated based on the one or more of the first to third reference values;
including methods. 2. The method of claim 1, wherein determining that the biosensor is contaminated is based on all of the first to third reference values. The first reference value includes the sum of the first current values for about 0.2 seconds to about 0.75 seconds after applying the first predetermined voltage, and the first reference value includes the sum of the first current values for about 0.2 seconds to about 0.75 seconds after the first predetermined voltage is applied, and the biosensor is contaminated. 2. The method of claim 1, wherein determining that the first reference value is greater than about 6.5 μA. 2. The method of claim 1, wherein the second reference value is based on the peak current value measured during the second predetermined time interval being less than about 12.5 μA. 5. The method of claim 4, wherein the peak current value is measured approximately 0.05 seconds after applying the second predetermined voltage. The third reference value is a current value measured approximately 0.1 seconds after applying the second predetermined voltage and the peak current value measured during the second predetermined time interval. 2. The method of claim 1, wherein the step of determining that the biosensor is contaminated is based on the third reference value being between about −3.5 μA and 0 μA. further comprising determining a fourth reference value based on the magnitude of the difference between the highest current value and the lowest current value measured during the first predetermined time interval, the biosensor being contaminated; 2. The method of claim 1, wherein the step of determining is further based on the fourth reference value. The fourth reference value is the difference between the highest current value and the lowest current value measured between about 0.2 seconds and about 0.75 seconds after applying the first predetermined voltage. 8. The method of claim 7, wherein the step of determining that the biosensor is contaminated is based on the measured second reference value being greater than about 0.57 μA. The step of determining a fifth reference value based on the minimum current value measured during the first predetermined time interval further comprises the step of determining that the biosensor is contaminated. The method according to claim 1, based on five reference values. The fifth reference value includes a minimum of the current value between about 0.2 seconds and about 0.75 seconds after applying the first predetermined voltage, and the biosensor is contaminated. 10. The method of claim 9, wherein the step of determining is based on the fifth criterion value being greater than about 0 μA. 2. The method of claim 1, further comprising calculating the analyte concentration based on the second current value and a third current value measured at a third predetermined time interval. Calculating the analyte concentration comprises:

including using equations of the form, where
G _basic is the analyte concentration;
i _r is the sum of the third current values during the third predetermined time interval,
i _l is the sum of the second current values during the second predetermined time interval,

in,
i _pc is the measured peak current value during said third predetermined time interval;
i _pb is the measured peak current value during said second predetermined time interval;
i _ss is the approximately steady state current value measured during the third predetermined time interval;
12. The method of claim 11, wherein a, b, p and z _gr are predetermined coefficients. a voltage source;
a user interface;
controller and
A test meter for determining the presence of contamination of a biosensor, comprising:
The controller,
applying a first predetermined voltage between spaced apart electrodes of an electrochemical cell with the voltage source for a first predetermined time interval;
measuring a first current value during the first predetermined time interval;
determining a first reference value based on the sum of the first current values during the first predetermined time interval;
applying a second predetermined voltage between the spaced apart electrodes during a second predetermined time interval after the first predetermined time interval;
measuring a second current value during the second predetermined time interval;
a second reference value based on a peak current value measured during the second predetermined time interval and a rate of change of the current value measured after the peak current value during the second predetermined time interval; a step of determining a third reference value based on;
Displaying on a user interface whether the biosensor is contaminated based on the one or more of the first to third reference values;
A test measuring instrument configured to perform 14. The test meter of claim 13, wherein the test meter is further configured to calculate an analyte concentration based on the second current value and the third current value. Calculating the analyte concentration comprises:

including using equations of the form, where
G _basic is the analyte concentration;
_ir is the sum of the third current values during the third predetermined time interval,
i _l is the sum of the second current values during the second predetermined time interval,

in,
i _pc is the measured peak current value during said third predetermined time interval;
i _pb is the measured peak current value during said second predetermined time interval;
i _ss is the approximately steady state current value measured during the third predetermined time interval;
15. The test meter of claim 14, wherein a, b, p and z _gr are predetermined coefficients. 14. The test meter of claim 13, wherein determining that the biosensor is contaminated is based on all of the first to third reference values. The first reference value includes the sum of the first current values for about 0.2 seconds to about 0.75 seconds after applying the first predetermined voltage, and the first reference value includes the sum of the first current values for about 0.2 seconds to about 0.75 seconds after the first predetermined voltage is applied, and the biosensor is contaminated. 14. The test meter of claim 13, wherein the step of determining that the first reference value is greater than about 6.5 μA. 14. The test meter of claim 13, wherein the second reference value is based on the peak current value measured during the second predetermined time interval being less than about 12.5 μA. The third reference value is a current value measured approximately 0.1 seconds after applying the second predetermined voltage and the peak current value measured during the second predetermined time interval. 14. The test meter of claim 13, wherein the step of determining that the biosensor is contaminated is based on the third reference value being between about −3.5 μA and 0 μA. .

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