CN1902477A

CN1902477A - Method of reducing interferences in an electrochemical sensor using two different applied potentials

Info

Publication number: CN1902477A
Application number: CN 200480039526
Authority: CN
Inventors: O·W·H·达维斯
Original assignee: LifeScan Scotland Ltd
Current assignee: LifeScan Scotland Ltd
Priority date: 2003-10-31
Filing date: 2004-10-29
Publication date: 2007-01-24
Anticipated expiration: 2024-10-29
Also published as: CN101493466A; CN1902478A; CN100473983C; CN101163963B; CN100473982C; CN1902479A; CN1902481A; CN101533007B; CN1902480A; CN101163963A; CN101493466B; CN101533007A

Abstract

The present invention is directed to an improved meter that utilizes a method of reducing the effects of interfering compounds in the measurement of analytes and more particularly to a method of reducing the effects of interfering compounds in a system wherein the test strip (600) utilizes two or more working electrodes. In the present invention, a meter is described which applies a first potential (E1) to a first working electrode (12) and a secon d potential (E2), having the same polarity but a greater magnitude than the first potential is applied to a second working electrode (14).

Description

Method for reducing interferences in an electrochemical sensor using two different applied potentials

Background

Electrochemical glucose test strips, such as in OneTouch available from LifeScan, inc^®Ultra^®Those electrochemical glucose test strips used in whole blood test kits are designed to measure the glucose concentration in a blood sample from a diabetic patient. The measurement of glucose is based on the specific oxidation of glucose by the flavoenzyme glucose oxidase. During this reaction, the enzyme is reduced. The enzyme is reoxidized by reaction with the mediator ferricyanide, which itself is reduced during the process or reaction. These reactions are summarized below.

When the reaction is carried out using a potential applied between two electrodes, an electric current can be generated by electrochemical reoxidation of the reduced mediator ions (ferrocyanide) at the electrode surface. Thus, since in an ideal environment the amount of ferrocyanide generated during the chemical reaction described above is proportional to the amount of glucose in the sample placed between the two electrodes, the current generated will be proportional to the glucose content of the sample. Redox mediators such as ferricyanide are compounds that exchange electrons between a redox enzyme such as glucose oxidase and an electrode. As the concentration of glucose in the sample increases, the amount of reduced mediator formed also increases, and thus there is a direct correlation between the current generated by reoxidation of the reduced mediator and the glucose concentration. In particular, electron transfer across the electrical interface results in the flow of current (2 moles of electrons per mole of glucose oxidized). Therefore, the current due to the introduction of glucose is referred to as a glucose current.

Because it can be very important to know the concentration of glucose in blood, particularly in the blood of diabetics, meters have been developed that use the above principles to be able to sample from the average person at any time and measure the glucose concentration. The resulting glucose current is monitored by the meter and converted to a reading of glucose concentration using a preset algorithm that relates current to glucose concentration by a simple mathematical formula. Typically, the meter works in conjunction with a disposable strip that includes, in addition to an enzyme (glucose oxidase) and a mediator (ferricyanide), a sample chamber and at least two electrodes disposed within the sample chamber. In use, a user sticks their finger or other convenient site to cause bleeding and introduces a blood sample into the sample chamber, thereby initiating the chemical reaction.

In electrochemical terms, the function of the meter is twofold. First, it provides a polarization voltage (for OneTouch)^®Ultra^®Approximately 0.4V) that polarizes the electrical interface and allows current to flow at the surface of the carbon working electrode. Secondly, it measures the current flowing in the external circuit between the anode (working electrode) and the cathode (reference electrode). Thus, the meter can be viewed as a simple electrochemical system that operates in a two-electrode mode, and in practice can use the 3 rd, or even the 4 th, electrode to assist in measuring glucose and/or other functions in the meter.

In most cases, the formula given above is considered to be a sufficient approximation of the chemical reaction that is performed on the test strip, and the meter reading is a sufficiently accurate representation of the glucose concentration of the blood sample. However, in some cases and for some purposes, it is advantageous to improve the accuracy of the measurement. For example, a portion of the current measured at the electrodes is due to other chemicals or compounds present in the sample. When such additional chemicals or compounds are present, they may be referred to as interfering compounds, and the resulting additional current may be referred to as interfering current.

Potential interfering chemicals (i.e., compounds found in physiological fluids such as blood that produce interfering currents in the presence of an electric field) include ascorbate, urate, and acetaminophen (Tylenol)^TMOr Paracetamol). In electrochemical meters (e.g., glucose meters) for measuring the concentration of an analyte in a physiological fluid, one mechanism for generating an interfering current involves the oxidation of one or more interfering compounds by reduction of an enzyme (e.g., glucose oxidase). In such meters, another mechanism for generating an interference current involves the oxidation of one or more interfering compounds by the reduction of a mediator (e.g., ferricyanide). In such meters, another mechanism for generating an interference current involves the oxidation of one or more interfering compounds at the working electrode. Thus, the total current measured at the working electrode is the superposition of the current due to oxidation of the analyte and the current due to oxidation of the interfering compound. The oxidation of interfering compounds may be the result of interaction with the enzyme, the mediator, or may occur directly at the working electrode.

In general, the potential interfering compound may be oxidized at the electrode surface and/or by a redox mediator. The oxidation of interfering compounds in the glucose measurement system causes the measured oxidation current to be related to both glucose and interfering compounds. Thus, if the concentration of interfering compounds is oxidized with the same efficiency as glucose and/or the interfering compound concentration is significantly higher than the glucose concentration, it may affect the measured glucose concentration.

The co-oxidation of an analyte (e.g. glucose) with an interfering compound is particularly problematic in the following cases: the standard potential of the interfering compound (i.e., the potential at which the compound is oxidized) is similar in magnitude to the standard potential of the redox mediator, resulting in a significant proportion of the interference current generated by the oxidation of the interfering compound at the working electrode. The current generated due to the oxidation of interfering compounds at the working electrode may be referred to as a direct interference current. Therefore, it would be advantageous to reduce or minimize the impact of direct interference currents on analyte concentration measurements. Previous methods of reducing or eliminating direct interference current include designing to prevent interfering compounds from reaching the working electrode, thereby reducing or eliminating the direct interference current generated by the compounds being eliminated.

One strategy to reduce the effect of interfering compounds that generate direct interfering currents is to place a negatively charged membrane on top of the working electrode. As an example, sulfonated fluoropolymers such as NAFION^TMPlaced over the working electrode to repel all negatively charged chemicals. In general, many interfering compounds, including ascorbate and urate, are negatively charged, and thus are repelled from oxidation when the surface of the working electrode is covered by a negatively charged membrane. However, since certain interfering compounds such as paracetamol are not negatively charged and thus can pass through the negatively charged membrane, the use of a negatively charged membrane will not eliminate direct interfering currents. Another disadvantage of covering the working electrode with a negatively charged membrane is that the commonly used redox mediators, such as ferricyanide, are negatively charged and cannot exchange electrons with the electrode through the membrane. A further disadvantage of using a negatively charged membrane on top of the working electrode is that the potential slows down the diffusion of the reduced mediator to the working electrode, thereby increasing the measurement time. Yet another disadvantage of using a negatively charged membrane over the working electrode is that it adds complexity and cost to the manufacture of test strips having a negatively charged membrane.

Another strategy that can be used to reduce the direct interference current is to place a size selective membrane on top of the working electrode. As an example, a 100 dalton size exclusion membrane, such as a cellulose acetate membrane, may be placed over the working electrode to exclude compounds with a molecular weight greater than 100 daltons. In this embodiment, an oxidoreductase enzyme, such as glucose oxidase, is placed on the size exclusion membrane. In the presence of glucose and oxygen, glucose oxidase produces hydrogen peroxide in an amount proportional to the glucose concentration. It should be noted that glucose and most redox mediators have molecular weights greater than 100 daltons and therefore cannot pass through a size selective membrane. However, hydrogen peroxide has a molecular weight of 34 daltons and is therefore capable of passing through size selective membranes. Typically, most compounds have a molecular weight greater than 100 daltons and are therefore excluded from oxidation at the electrode surface. Because some interfering compounds have a small molecular weight and are therefore capable of passing through the size selective membrane, the use of a size selective membrane will not eliminate direct interfering currents. Another disadvantage of using a size selective membrane over the working electrode increases the manufacturing complexity and cost of test strips with size selective membranes.

Another strategy that can be used to reduce the effects of direct interference current is to use a redox mediator with a low redox potential, for example a redox mediator with a redox potential of about-300 mV to +100mV (vs saturated calomel electrode). This enables a lower potential to be applied to the working electrode, thereby reducing the rate at which interfering compounds are oxidized by the working electrode. Examples of redox mediators having a lower redox potential include osmium bipyridyl complexes, ferrocene derivatives, and quinone derivatives. However, redox mediators with lower redox potentials are often difficult to synthesize, less stable and less soluble.

Another strategy that can be used to reduce the effect of interfering compounds is to use a dummy electrode in combination with a working electrode. The current measured at the dummy electrode can then be subtracted from the current measured at the working electrode to compensate for the effects of interfering compounds. If the dummy electrode is bare (i.e., not covered by the enzyme or mediator), the current measured at the dummy electrode will be directly proportional to the direct interference current, and subtracting the current measured at the dummy electrode from the current measured at the working electrode will reduce or eliminate the effect of direct oxidation of the interfering compound at the working electrode. If the dummy electrode is covered with a redox mediator, the current measured at the dummy electrode will be a combination of the direct interference current and the interference current due to the reduction of the redox mediator by the interfering compound. Thus, subtracting the current measured at the dummy electrode covered with redox mediator from the current measured at the working electrode will reduce or eliminate the effect of direct oxidation of interfering compounds and the effect of interference at the working electrode due to reduction of the redox mediator by the interfering compounds. In some cases, the dummy electrode may also be covered with an inert protein or deactivated redox enzyme to mimic the effect of redox mediators and enzymes on diffusion. Because the test strip preferably has a small sample chamber so that a diabetic patient does not need to give a large blood sample, the inclusion of additional electrodes that increase the volume of the sample chamber relative to the volume of the sample chamber when no additional electrodes are used to measure an analyte (e.g., glucose) may be disadvantageous. Furthermore, it may be difficult to directly correlate the current measured at the dummy electrode with the interference current measured at the working electrode. Finally, because the dummy electrode may be covered with a material (e.g., redox mediator) that is different from the material used to cover the working electrode (e.g., redox mediator and enzyme), a test strip that uses the dummy electrode as a means to reduce or eliminate the effects of interfering compounds in a multiple working electrode system may increase the cost and complexity of manufacturing the test strip.

Measuring analytes using multiple working electrodes, e.g. in OneTouch^®Ultra^®Some test strip designs of the systems used in the measurement system are advantageous because two working electrodes are used. In such systems, it would therefore be advantageous to develop methods to reduce or eliminate the effects of interfering compounds. More particularly, it would be advantageous to develop a method of reducing or eliminating the effects of interfering compounds without the use of dummy electrodes, intermediate thin films, or redox mediators having a low redox potential.

Summary of The Invention

The present invention relates to methods of reducing the effects of interfering compounds in the measurement of analytes, and more particularly, to methods of reducing the effects of interfering compounds in systems in which a test strip uses two or more working electrodes. In one embodiment of the invention, a first potential is applied to a first working electrode and a second potential, of the same polarity as the first potential but greater magnitude than the first potential, is applied to a second working electrode. For embodiments in which a reduction current is used to measure the analyte concentration, the second potential may also be of a smaller magnitude than the first potential. In one embodiment, the first working electrode and the second working electrode may be coated with an analyte-specific enzymatic reagent and a redox mediator. The first potential applied to the first working electrode is selected to be sufficient to oxidize the reduced redox mediator in a diffusion limited manner, while the second potential is selected to be greater in magnitude (i.e., absolute value) than the first potential, thereby allowing more efficient oxidation to occur at the second working electrode. In this embodiment of the invention, the current measured at the first working electrode comprises the analyte current and the interfering compound current, and the current measured at the second working electrode comprises the analyte overpotential current and the interfering compound overpotential current. It should be noted that both the analyte current and the analyte overpotential current refer to currents corresponding to the analyte concentration, and that the currents are the result of oxidation of the reduced mediator. In one embodiment of the present invention, the relationship between the current at the first working electrode and the current at the second working electrode can be defined by the following equation,

A_{1} = \frac{W_{2} - {YW}_{1}}{X - Y}

wherein A is₁Is the analyte current at the first working electrode, W₁Is at the first working electrodeMeasured current, W₂Is the current measured at the second working electrode,x is an analyte-dependent voltage effect factor and Y is an interfering compound-dependent voltage effect factor. Using the above formula in the method of the present invention, it is possible to reduce the effect of oxidation current due to the presence of interfering compounds and to calculate a corrected current value that is more representative of the concentration of the analyte in the sample being tested.

In one embodiment of the present invention, the glucose concentration in the sample placed on the test strip may be calculated as follows: the sample is placed on a test strip having first and second working electrodes and a reference electrode, at least the first and second working electrodes being covered by a compound (e.g., an enzyme and a redox mediator) adapted to promote oxidation of glucose and transfer of electrons from the oxidized glucose to the first and second working electrodes when a potential is applied between the first working electrode and the reference electrode and between the second working electrode and the reference electrode. According to the invention, a first potential is applied between the first working electrode and the reference electrode, the magnitude of the first potential being selected to be sufficient to ensure that the magnitude of the current generated by oxidation of glucose in the sample is limited only by factors other than the applied voltage (e.g. diffusion). According to the invention, a second potential is applied between the second working electrode and the reference electrode, the second potential being greater in magnitude than the first potential, and in one embodiment of the invention, the second potential is selected to increase oxidation of the interfering compound at the second working electrode. In another embodiment of the invention, the following formula may be used to reduce the effect of the oxidation current due to the presence of interfering compounds on the current used to calculate the glucose concentration in the sample. In particular, the calculated current A may be used_1GTo derive the glucose concentration, wherein

A_{1 G} = \frac{W_{2} - {YW}_{1}}{X_{G} - Y}

Wherein A is_1GIs a glucose current, W₁Is the current measured at the first working electrode, W₂Is the current, X, measured at the second working electrode_GIs a glucose-dependent voltage effector, and Y is an interfering compound-dependent voltage effector.

Brief Description of Drawings

The novel features of the invention are set forth with particularity in the appended claims. A better understanding of the features and advantages of the present invention will be obtained by reference to the following detailed description that sets forth illustrative embodiments, in which the principles of the invention are utilized, and the accompanying drawings of which:

FIG. 1 is an exploded perspective view of an embodiment of a test strip for use in the present invention.

FIG. 2 is a diagrammatic view of a meter and test strip for use with the present invention.

Fig. 3 is a hydrodynamic voltammogram showing the dependence between applied voltage and measured current.

Detailed Description

While the present invention is particularly suited for measuring glucose concentrations in blood, it will be apparent to those skilled in the art that the methods described in the present invention may be adapted to improve the selectivity of other systems for electrochemical measurement of analytes in physiological fluids. Examples of systems that may be adapted to use the methods of the present invention to increase selectivity include electrochemical sensors for measuring the concentration of lactate, alcohol, cholesterol, amino acids, choline, and fructosamine in physiological fluids. Examples of physiological fluids that may contain such analytes include blood, plasma, serum, urine, and interstitial fluid. It should be further understood that while the method of the present invention is described in an electrochemical system in which the measured current is produced by oxidation, the present invention is equally applicable to systems in which the measured current is produced by reduction.

The present invention relates to a method for improving the selectivity of an electrochemical measurement system, which is particularly suitable for use in blood glucose measurement systems. More particularly, the present invention relates to a method of improving the selectivity of a blood glucose measurement system by partially or fully correcting for the effects of direct interference currents. In such systems, selectivity is the ability of the system to accurately measure the concentration of glucose in a sample of physiological fluid that includes one or more compounds that can produce interfering currents. Thus, the increased selectivity reduces the current generated at the working electrode due to the presence of interfering compounds (i.e., compounds other than glucose that oxidize to produce an interfering current) and makes the measured current more representative of the glucose concentration. In particular, the measured current may be a function of the oxidation of interfering compounds commonly found in physiological fluids, such as paracetamol (Tylenol)^TMOr Paracetamol), ascorbic acid, bilirubin, dopamine, gentisic acid, glutathione, levodopa, methyldopa, tolazamide, tolbutamide and uric acid. Such interfering compounds may be oxidized, for example, by chemical reaction with a redox mediator, or by oxidation at the surface of an electrode.

In a fully selective system, there will be no oxidation current generated by any interfering compounds, and the entire oxidation current will be generated by glucose oxidation. However, if the oxidation of interfering compounds and the resulting oxidation current cannot be avoided, the present invention describes a method of eliminating some or all of the effects of interfering compounds by quantitatively determining the proportion of the oxidation current produced by the interfering compounds to the total oxidation current and subtracting that amount of current from the total oxidation current. In particular, in the methods of the invention, a test strip comprising a first working electrode and a second working electrode is used, two different potentials are applied, and the oxidation current generated at each working electrode is measured to estimate the ratio of oxidation current occupied by each of glucose and interfering compounds.

In one embodiment of the method of the present invention, the test strip used comprises a sample chamber containing a first working electrode, a second working electrode, and a reference electrode. The first working electrode, the second working electrode, and the reference electrode are covered with glucose oxidase (enzyme) and ferricyanide (redox mediator). When a blood sample (physiological fluid) is placed in the sample chamber, the glucose oxidase is reduced by the glucose in the blood sample, producing gluconic acid. Gluconic acid is then oxidized by reduction of ferricyanide to ferrocyanide, producing a reduced redox mediator whose concentration is proportional to the glucose concentration. An example of a test strip that can be suitable for use in the method of the present invention is OneTouch sold by LifeScan, inc^®Ultra^®And (3) testing the strip. Other test strips are described in International publications WO 01/67099A1 and WO01/73124A 2.

In one embodiment of the method of the present invention, a first potential is applied to a first working electrode and a second potential is applied to a second working electrode. In this embodiment, the magnitude of the first potential is selected such that the glucose current response is relatively insensitive to the applied potential, such that the magnitude of the glucose current at the first working electrode is limited by the amount of reduced redox mediator that diffuses to the first working electrode. It should be noted that glucose is not directly oxidized at the working electrode, but is indirectly oxidized through the use of a redox enzyme and a redox mediator. In the context of the present invention, glucose current refers to the oxidation of a reduced redox mediator in relation to glucose concentration. In embodiments of the invention where ferricyanide/ferrocyanide is the redox mediator and carbon is the working electrode, the first potential can be from about 0 millivolts to about 500 millivolts, more preferably from about 385 millivolts to about 415 millivolts, and even more preferably about 395-405 mV. A second potential is applied to the second working electrode such that the second potential is greater than the first potential. Wherein the applied potential is greater than the potential required to oxidize glucose. In one embodiment of the invention, when ferricyanide/ferrocyanide is the redox mediator and carbon is the working electrode, the second potential can be from about 50 millivolts to about 1000 millivolts, more preferably from about 420 millivolts to about 1000 millivolts, and even more preferably about 395-405 mV.

Because the glucose current does not increase or only minimally increases with increasing potential, the glucose current at the second working electrode should be substantially equal to the glucose current at the first working electrode, even though the potential at the second working electrode is greater than the potential at the first working electrode. Thus, any additional current measured at the second working electrode may be attributed to oxidation of interfering compounds. In other words, a higher potential at the second working electrode should cause the glucose overpotential current measured at the second working electrode to be equal or substantially equal in magnitude to the glucose current at the first working electrode, because the first and second potentials are in a limited glucose current range that is insensitive to changes in the applied potential. However, in practice, other parameters may affect the measured current, for example, when a higher potential is applied to the second working electrode, the total current at the second working electrode often increases slightly as a result of IR drop or capacitive effects. When there is an IR drop (i.e., uncompensated resistance) in the system, the higher applied potential causes the measured current to increase. An example of an IR drop can be a nominal resistance of the first working electrode, the second working electrode, the reference electrode, the physiological fluid between the working electrode and the reference electrode. In addition, applying a higher potential results in the formation of a larger ionic bilayer that is formed at the electrode/liquid interface, increasing the ionic capacitance and resulting current at the first working electrode or the second working electrode.

To determine the actual relationship between the glucose current measured at the first working electrode and the glucose current measured at the second working electrode, a suitable formula must be developed. It should be noted that the glucose current at the second working electrode may also be referred to as a glucose overpotential current. The proportional relationship between the glucose current and the glucose overpotential current can be described by the following equation.

X_G×A_1G＝A_2G(formula 1)

Wherein X_GIs a glucose-dependent voltage effector, A_1GIs the glucose current at the first working electrode, A_2GIs the glucose current at the second working electrode.

In one embodiment of the invention, when ferricyanide/ferrocyanide is the redox mediator and carbon is the working electrode, the voltage effector can be expected to be about 0.95 to about 1.1 for glucose. In this embodiment of the invention, the higher potential has no significant effect on the glucose oxidation current because the redox mediator (ferrocyanide) has fast electron transfer kinetics and reversible electron transfer characteristics with the working electrode. Since the glucose current does not increase with increasing potential after a certain point, it can be said that the glucose current is saturated or in the case of diffusion limitation.

In the above-described embodiments of the present invention, glucose is indirectly measured by oxidizing ferrocyanide at the working electrode, and the ferrocyanide concentration is directly proportional to the glucose concentration. For a particular electrochemical compound, the standard potential (E °) value is a measure of the ability of the compound to exchange electrons with other compounds. In the method of the invention, the potential at the first working electrode is selected to be greater than the standard potential (E) of the redox mediator. Because the first potential is selected to be sufficiently greater than the E value of the redox couple, the rate of oxidation does not increase significantly with increasing applied potential. Thus, applying a larger potential at the second working electrode will not increase oxidation at the second working electrode, and any increased current measured at the higher potential electrode must be due to other factors such as oxidation of interfering compounds.

Fig. 3 is a hydrodynamic voltammogram showing the dependence between applied voltage and measured current, where ferricyanide/ferrocyanide is the redox mediator and carbon is the working electrode. Each data point on the graph represents at least one experiment in which the current was measured 5 seconds after the voltage was applied between the working electrode and the reference electrode. Figure 3 shows that at about 400mV, current forms the beginning of the plateau region because the applied voltage is sufficiently greater than the E ° value of ferrocyanide. Thus, as shown in fig. 3, when the potential reaches about 400mV, the glucose current becomes saturated because the oxidation of ferrocyanide is diffusion limited (the diffusion of ferrocyanide to the working electrode limits the magnitude of the measured current and is not limited by the electron transfer rate between ferrocyanide and the electrode).

In general, the current generated by oxidation of an interfering compound is not saturated by an increase in applied voltage, and exhibits much stronger dependence on the applied potential than the current generated by oxidation of ferrocyanide (which has been generated by the interaction of glucose with an enzyme and an enzyme with ferrocyanide). Interfering compounds generally have slower electron transfer kinetics than redox mediators (i.e., ferrocyanide). The reason for this difference is the fact that most interfering compounds undergo the inner sphere electron transfer pathway, whereas ferrocyanide undergoes the faster outer sphere electron transfer pathway. Typical internal sphere electron transfer pathways require a chemical reaction, such as hydride transfer, to occur before transferring electrons. In contrast, the outer sphere electron transfer pathway does not require a chemical reaction prior to transferring electrons. Therefore, the inner sphere electron transfer speed is generally slower than the outer sphere electron transfer because they require an additional chemical reaction step. Oxidation of ascorbic acid to dehydroascorbic acid is an example of internal sphere oxidation, which requires the release of two hydride moieties. The oxidation of ferricyanide to ferrocyanide is an example of outer sphere electron transfer. Thus, the current generated by interfering compounds typically increases when tested at higher potentials.

The relationship between the interfering compound current at the first working electrode and the overpotential current of the interfering compound at the second working electrode can be described by the following equation,

Y×I₁＝I₂(formula 2)

Wherein Y is an interfering compound-dependent voltage effector, I₁Is an interfering compound current, and I₂Is an interfering compound overpotential current. Because the interfering compound-dependent voltage effect factor Y depends on a variety of factors, including the particular interfering compound and all materials of the working electrode, the calculation of a particular interfering compound-dependent voltage effect factor may require experimentation to optimize the voltage effect factor for these criteria for a particular system, test strip, analyte, and interfering compound. Alternatively, under some conditions, the appropriate voltage effect factor may be derived or described mathematically.

In one embodiment of the invention where ferricyanide/ferrocyanide is the redox mediator and carbon is the working electrode, the interfering compound-dependent voltage effect factor Y may be used with respect to I₁And I₂The Tafel formula of (a) is described mathematically,

I_{1} = a^{'} \exp (\frac{η_{1}}{b^{'}})

(formula 2a)

I_{2} = a^{'} \exp (\frac{η_{2}}{b^{'}})

(formula 2b)

η therein₁＝E₁-E°，η₂＝E₂E, b' depends on the constants of the particular electroactive interfering compounds, E₁Is a first potential, and E₂The value of E (the standard potential of a particular interfering compound) is not important because it is offset in the calculation of a η, equations 2, 2a, 2b can be combined and rearranged, resulting in the following equations,

Y = \exp (\frac{Δη}{b^{'}})

(formula 2c)

Wherein Δ η ═ E₁-E₂Equation 2c provides a mathematical relationship describing the relationship between Δ η (i.e., the difference between the first potential and the second potential) and the interfering compound-dependent voltage effect factor YFrom about 1 to about 10. In one embodiment of the invention, the interfering compound-dependent voltage effect factor Y can be determined experimentally for a particular interfering compound or combination of interfering compounds. It should be noted that for interfering compounds, the interfering compound-dependent voltage effector Y is generally greater than the voltage effector X of glucose_G. As described in the following section, a) interfering compound current I₁Overpotential current I of interfering compound₂A mathematical relationship therebetween; and b) glucose Current A_1GWith glucoseGlucose overpotential current A_2GThe mathematical relationship between them enables the proposal of a glucose algorithm that reduces the effect of interfering compounds on the glucose measurement.

In one embodiment of the invention, an algorithm is developed to calculate a corrected glucose current (i.e., A) that is not affected by the interferent_1GAnd A_2G). After applying the sample to the test strip, a first potential is applied to the first working electrode and a second potential is applied to the second working electrode. At the first working electrode, a first current is measured, which can be described by the following equation,

W₁＝A_1G+I₁(formula 3)

Wherein W₁Is the first current at the first working electrode. In other words, the first current comprises the glucose current A_1GWith interference compound current I₁And (3) superposition. More specifically, the interfering compound current may be a direct interfering current as described above. At the second working electrode, a second current at a second potential or overpotential is measured, which can be described by the following equation,

W₂＝A_2G+I₂(formula 4)

Wherein W₂Is a second current at a second working electrode, A_2GIs a glucose overpotential current, I, measured at a second potential₂Is the overpotential current of the interfering compound measured at the second potential. More specifically, the interfering compound overpotential current may be the direct interfering compound current described above. The use includes 4 unknowns (A)_1G、A_2G、I₁And I₂) The 4 equations described above (equations 1-4), a corrected glucose current equation that is not affected by the interfering compound can be calculated.

As a first step in the derivation, A2G from equation 1 and I2 from equation 2 may be substituted into equation 4, resulting in equation 5 below.

W₂＝X_GA_1G+YI₁(formula 5)

Next, formula 3 is multiplied by the interfering compound-dependent voltage effect factor Y of the interfering compound to obtain formula 6.

YW₁＝YA_1G+TI₁(formula 6)

Subtracting equation 6 from equation 5 yields the following form as shown in equation 7

W₂-YW₁＝X_GA_1G-YA_1G(formula 7)

Equation 7 is rearranged to solve for the corrected glucose current A measured at the first potential_1GAs shown in equation 8.

A_{1 G} = \frac{W_{2} - {YW}_{1}}{X_{G} - Y}

(formula 8)

The corrected glucose current A is obtained by equation 8_1GWhich eliminates the effects of interference, which requires only the output currents of the first working electrode and the second working electrode (e.g., W)₁And W₂) Glucose dependent voltage effect factor X_GAnd an interfering compound-dependent voltage effect factor Y of the interfering compound.

Electrically connecting a glucose meter including electronic components to a glucose test strip to measure glucose from W₁And W₂The current is measured. In one embodiment of the present invention, X may be_GAnd Y is programmed into the glucose meter so that only the memory is read. In another embodiment of the invention, Y may be communicated to the meter by a calibration code chip. The correction code chip has a specific set of X-related bits in its memory_GAnd Y, which can be corrected for many specific test strips. This may explain the possibility of X_GAnd the lot-to-lot variation of test strips that occurred in Y.

In another embodiment of the present invention, the corrected glucose current in equation 8 may be used by the meter only if some threshold is exceeded. For example, if W₂Ratio W₁If it is 10% or more than 10%, the meter will use equation 8 to correct the output current. However, if W₂Ratio W₁If the concentration of the interfering compound is 10% or less, the concentration of the interfering compound is very low, so that the measuring instrument can simply take W₁And W₂To improve the accuracy and precision of the measurement. In order to replace the simple application of current W₁And W₂On average, a more accurate approach may be to use W₂/X_GTo average W₁Wherein a glucose-dependent voltage effect factor X is taken into account_G(Note)When I comes₂Very low, W according to

equations

1 and 4₂/X_GApproximately equal to A_1G). Using the strategy of equation 8 only in some cases where significant levels of interfering compounds are present in the sample mitigates the risk of overcorrection of the measured glucose current. It should be noted that when W₂Ratio W₁When sufficiently large (e.g., about 100% or more larger), this is indicative of a very high concentration of interfering compounds. In such a case, it may be desirable to output an error message instead of a glucose value, since very high levels of interfering compounds may cause the accuracy of equation 8 to be broken.

The following section will describe possible test strip embodiments that can be used with the proposed algorithm of the present invention shown in equation 8. Fig. 1 is an exploded perspective view of a test strip 600 that includes 6 layers disposed on a substrate 5. These 6 layers are conductive layer 50, insulating layer 16, reagent layer 22, adhesive layer 60, hydrophilic layer 70, and top layer 80. Test strip 600 may be manufactured in a series of steps in which conductive layer 50, insulating layer 16, reagent layer 22, adhesive layer 60 are disposed on substrate 5 using, for example, a screen printing process. Hydrophilic layer 70 and top layer 80 may be removed from a roll stock and laminated to substrate 5. The fully assembled test strip forms a sample receiving chamber that can receive a blood sample so that the blood sample can be analyzed.

Conductive layer 50 includes reference electrode 10, first working electrode 12, second working electrode 14, first contact 13, second contact 15, reference contact 11, and test strip detection plate 17. Suitable materials that can be used to form the conductive layer are Au, Pd, Ir, Pt, Rh, stainless steel, doped tin oxide, carbon, and the like. Preferably, the material for the conductive layer may be a carbon ink (carbon ink) such as those described in US 5653918.

Insulating layer 16 includes a cut-out (cutoff) 18 that exposes a portion of reference electrode 10, first working electrode 12, and second working electrode 14 that can be wetted by the liquid sample. As a non-limiting example, the insulating layer (16 or 160) may be Ercon E6110-116 Jet Black insulating Ink available from Ercon, Inc.

Reagent layer 22 may be disposed on a portion of conductive layer 50 and insulating layer 16. In one embodiment of the present invention, reagent layer 22 may include chemicals such as oxidoreductases and redox mediators that selectively react with glucose. During this reaction, a proportional amount of reduced redox mediator may be produced, which may be measured electrochemically, so that the glucose concentration may be calculated. Examples of reagent formulations or inks suitable for use in the present invention can be found in US patents 5,708,247 and 6,046,051; published International applications WO01/67099 and WO01/73124, both of which are incorporated herein by reference.

The adhesive layer 60 includes a first adhesive pad 24, a second adhesive pad 26, and a third adhesive pad 28. The side edges of first adhesive pad 24 and second adhesive pad 26 adjacent to reagent layer 22 each define a wall of the sample-receiving chamber. In one embodiment of the invention, the adhesive layer may comprise a water-based acrylic copolymer pressure sensitive adhesive commercially available from Tapespecialties LTD in Tring, Herts, United Kingdom (part & num; A6435).

Hydrophilic layer 70 includes a distal hydrophilic pad 32 and a proximal hydrophilic pad 34. As a non-limiting example, hydrophilic layer 70 may be a polyester having one hydrophilic surface, such as an anti-fog coating, commercially available from 3M. It should be noted that both the distal hydrophilic membrane 32 and the proximal hydrophilic membrane 34 are transparent, thereby enabling the user to view the liquid sample filling the sample-receiving chamber.

Top layer 80 includes transparent portion 36 and opaque portion 38. Top layer 80 is disposed on and bonded to hydrophilic layer 70. By way of non-limiting example, the top layer 40 may be polyester. It should be noted that transparent portion 36 substantially overlaps proximal hydrophilic pad 32, which enables the user to visually verify that the sample receiving chamber is sufficiently filled. The opaque portion 38 helps the user to observe the high contrast between the colored fluid, e.g., blood, in the sample receiving chamber and the opaque region of the top film.

Fig. 2 is a simplified schematic diagram illustrating a meter 500 coupled to a test strip 600. Meter 500 has 3 electrical contacts that make electrical connections to first working electrode 12, second working electrode 14, and reference electrode 10. In particular, connector 101 connects voltage source 103 to first working electrode 12, connector 102 connects voltage source 104 to second working electrode 14, and common connector 100 connects

voltage sources

103 and 104 to reference electrode 10. When performing a test, voltage source 103 in meter 500 applies a first potential E between first working electrode 12 and reference electrode 10₁ Voltage source 104 applies a second potential E between second working electrode 14 and reference electrode 10₂. A blood sample is applied such that first working electrode 12, second working electrode 14, and reference electrode 10 are covered with blood. This causes reagent layer 22 to hydrate, producing ferrocyanide in an amount proportional to the concentration of glucose and/or interfering compounds present in the sample. 5 seconds after sample application, the meter 500 measures the first working electrode 12 and second working electrode 14.

In the first and second test strip embodiments described above, first working electrode 12 and second working electrode 14 have the same area. It should be noted that the present invention is not limited to test strips having the same area. For the above-described test strip embodiments in which the areas are different, the output current of each working electrode must be normalized to the area. Because the output current is proportional to the area, the terms in equations 1-8 can be expressed in amperes of potential (current) or amperes per potential area (i.e., current density).

It should be recognized that equivalent structures may be substituted for the structures illustrated and described herein, and that the described embodiments of the invention are not the only structures that may be used in the present invention. Further, it should be understood that each of the above-described structures has a function, and such a structure may be referred to as a means for performing that function. While preferred embodiments of the present invention have been shown and described, it will be obvious to those skilled in the art that such embodiments are provided by way of example only. Numerous variations, changes, and substitutions will now occur to those skilled in the art without departing from the invention. It should be understood that various alternatives to the embodiments of the invention described herein may be employed in practicing the invention. It is intended that the following claims define the scope of the invention and that methods and structures within the scope of these claims and their equivalents be covered thereby.

Claims

1. A method of reducing interference in an electrochemical sensor, the method comprising:

applying a first potential to the first working electrode;

applying a second potential to a second working electrode, wherein said second potential is greater than the absolute value of said first potential;

measuring a first current at the first working electrode, the first current comprising an analyte current and an interfering compound current;

measuring a second current at said second working electrode, said second current comprising an analyte overpotential current and an interfering compound overpotential current, wherein said analyte overpotential current has a first proportional relationship to said analyte current, and wherein said interfering compound overpotential current has a second proportional relationship to said interfering compound current; and

calculating a corrected current value representative of the analyte concentration using an equation that is a function of the first current, the second current, the first direct relationship, and the second direct relationship.

2. The method of claim 1, wherein the formula is

A_{1} = \frac{W_{2} - {YW}_{1}}{X - Y}

Wherein A is₁Is the analyte current, W₁Is said first current, W₂Is the second current, X is an analyte voltage effect factor, and Y is an interfering compound voltage effect factor.

3. The method of claim 1, wherein the analyte is glucose.

4. The method of claim 1, wherein the first potential is about 385 mv to about 415 mv for the electrochemical sensor comprising a carbon working electrode and a ferrocyanide redox mediator.

5. The method of claim 1, wherein said second potential is between about 420 millivolts and about 1000 millivolts for said electrochemical sensor comprising a carbon working electrode and a ferrocyanide redox mediator.

6. The method of claim 1, wherein the interfering compound current is generated as a result of oxidation of at least one compound selected from the group consisting of: paracetamol, ascorbic acid, bilirubin, dopamine, gentisic acid, glutathione, levodopa, methyldopa, tolazamide, tolbutamide and uric acid.

7. The method of claim 1, wherein said first proportional relationship is

XxA₁＝A₂

Wherein X is the analyte voltage effector, A₁Is the analyte current, and A₂Is the analyte overpotential current.

8. The method of claim 1, wherein said second proportional relationship is

YxI₁＝I₂

Wherein Y is the voltage effector of the interfering compound, I₁Is the interfering compound current, and I₂Is the overpotential current of the interfering compound.