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

Abstract

The present invention relates to a method of reducing the effect of interfering compounds in the measurement of an analyte, and more particularly, to a method of reducing the effect of interfering compounds in a system wherein the test strip (600) uses two or more working electrodes (12, 14). In the present invention, a first potential (E1) is applied to the first working electrode (12), and a second potential (E1) having the same polarity but greater than the first potential (E1) is applied to the second working electrode (14). potential (E2).

Description

Method for Reducing Interference in Electrochemical Sensors Using Two Different Applied Potentials

Background of the Invention

Electrochemical glucose test strips, such as those used in the ^{OneTouch® Ultra®} Whole Blood Test Kit from LifeScan, Inc., are designed to measure the concentration of glucose in blood samples from diabetic patients. The measurement of glucose is based on the specific oxidation of glucose by flavin enzyme-glucose oxidase. During this reaction, the enzyme is reduced. By reacting with the mediator ferricyanide, the enzyme is reoxidized, while during this process or reaction ferricyanide itself is reduced. These responses are summarized below.

      

    

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

Because it may be very important to know the concentration of glucose in the blood, especially in diabetics, a measurement using the above principle to be able to take a sample from a normal person at any time and measure the glucose concentration has been developed. instrument. The resulting glucose current is monitored by the meter and converted to a reading of glucose concentration using a pre-set algorithm that relates current to glucose concentration through a simple mathematical formula. Typically, the meter works with a disposable strip comprising, in addition to the enzyme (glucose oxidase) and mediator (ferricyanide), a sample chamber and at least two electrodes placed within the sample chamber. In use, the user pricks their finger or other convenient location to induce bleeding and introduces a blood sample into the sample chamber, thereby initiating the chemical reaction described above.

In electrochemical terms, the function of the meter is twofold. First, it provides a polarizing voltage (approximately 0.4V for the OneTouch ^® Ultra ^® ) that polarizes the electrical interface and allows current to flow across the surface of the carbon working electrode. Second, it measures the current flowing in an external circuit between the anode (working electrode) and cathode (reference electrode). The meter can therefore be viewed as a simple electrochemical system that operates in a two-electrode mode, where in practice a 3rd, or even a 4th electrode can also be used to help measure glucose and/or in the meter or perform other functions.

In most cases, the formula given above is considered to be a sufficient approximation of the chemical reaction taking place on the test strip, and the reading of the meter 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 increase the accuracy of the measurements. 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 an interfering current.

Potential-disturbing chemicals (ie, compounds found in physiological fluids such as blood that produce disturbing electrical currents in the presence of an electric field) include ascorbate, urate, and paracetamol (Tylenol ^™ or Paracetamol). In electrochemical meters (such as glucose meters) used to measure the concentration of analytes in physiological fluids, one mechanism for generating interfering currents involves the reduction of one or more interfering compounds by enzymes (such as glucose oxidase). oxidation. In such meters, another mechanism for generating interfering currents involves oxidation of one or more interfering compounds through reduction of a mediator (eg, ferricyanide). In such meters, another mechanism for generating interfering currents involves oxidation of one or more interfering compounds at the working electrode. Therefore, the total current measured at the working electrode is the superposition of the current due to the oxidation of the analyte and the current due to the oxidation of the interfering compound. Oxidation of interfering compounds can be the result of interactions with enzymes, mediators or can occur directly at the working electrode.

In general, potential-interfering compounds can be oxidized at the electrode surface and/or via redox mediators. Oxidation of the interfering compound in the glucose measuring system causes the measured oxidation current to be related both to glucose and to the interfering compound. Therefore, if the concentration of the interfering compound oxidizes with the same efficiency as glucose and/or the concentration of the interfering compound is significantly higher than the glucose concentration, it can affect the measured glucose concentration.

Co-oxidation of an analyte (e.g., glucose) with an interfering compound is particularly problematic when the standard potential of the interfering compound (i.e., the potential at which the compound is oxidized) is similar in magnitude to that of the redox mediator, resulting in The interference current generated by the oxidation of interfering compounds on the working electrode accounts for a considerable proportion. The current due to the oxidation of the interfering compound at the working electrode can be referred to as the direct interfering current. Therefore, it would be advantageous to reduce or minimize the effect of direct interfering currents on the measurement of analyte concentration. Previous approaches to reducing or eliminating direct interfering currents have involved designing devices that prevent interfering compounds from reaching the working electrode, thereby reducing or eliminating direct interfering currents produced by the eliminated compounds.

One strategy to reduce the influence of interfering compounds that produce direct interfering currents is to place a negatively charged thin film on top of the working electrode. As an example, a sulfonated fluoropolymer such as NAFION ^™ can be placed over the working electrode to repel any negatively charged chemicals. In general, many interfering compounds, including ascorbate and urate, are negatively charged, so when the surface of the working electrode is covered with a negatively charged film, these interfering compounds are repelled from oxidation. However, since some interfering compounds such as paracetamol are not negatively charged and thus can pass through negatively charged membranes, the use of negatively charged membranes will not eliminate the direct interfering currents. Another disadvantage of covering the working electrode with a negatively charged film is that commonly used redox mediators such as ferricyanide are negatively charged and cannot exchange electrons with the electrode through the film. A further disadvantage of using a negatively charged film 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 film on top of the working electrode is the increased complexity and cost of producing test strips with negatively charged films.

Another strategy that can be used to reduce direct interference currents 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, can be placed on the working electrode to exclude compounds with molecular weights greater than 100 Daltons. In this embodiment, an oxidoreductase, such as glucose oxidase, is placed on a size exclusion membrane. In the presence of glucose and oxygen, glucose oxidase produces hydrogen peroxide in an amount proportional to the concentration of glucose. It should be noted that glucose and most redox mediators have a molecular weight greater than 100 Daltons and thus cannot select films by size. However, hydrogen peroxide has a molecular weight of 34 Daltons, thus enabling film selection by size. In general, most compounds have a molecular weight greater than 100 Daltons and are therefore excluded from being oxidized on the electrode surface. Because some interfering compounds have small molecular weights and are therefore able to pass through the size-selective membrane, the use of a size-selective membrane will not eliminate the direct interfering current. Another disadvantage of using a size selective membrane over the working electrode increases the complexity and cost of manufacturing test strips with the size selective membrane.

Another strategy that can be used to reduce the impact of direct interfering currents is to use redox mediators with low redox potentials, eg redox mediators with redox potentials of about -300 mV to +100 mV (vs saturated calomel electrodes). 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 effect of interfering compounds. If the dummy electrode is bare (that is, not covered by enzyme or mediator), the current measured at the dummy electrode will be proportional to the direct interfering current, and the current measured at the dummy electrode will be subtracted from the current measured at the working electrode. Going will reduce or eliminate the effect of direct oxidation of interfering compounds on 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 interfering current and the interfering current due to the reduction of the redox mediator by the interfering compound. Therefore, subtracting the current measured at the dummy electrode covered with a redox mediator from the current measured at the working electrode will reduce or eliminate the effect of direct oxidation of the interfering compound and the reduction of the redox mediator due to the interfering compound at the working electrode. The effect of interference caused by reduction. In some cases, pseudoelectrodes can also be covered with inert proteins or inactivated redox enzymes to mimic the role of redox mediators and enzymes on diffusion. Since the test strip preferably has a small sample chamber so that the diabetic does not need to give a large blood sample, the inclusion of the additional electrodes results in an increase in the sample chamber volume relative to the sample chamber volume if no additional electrodes were used to measure the analyte (eg glucose) May be disadvantageous. Furthermore, it may be difficult to directly correlate the current measured at the dummy electrode with the interfering current measured at the working electrode. Finally, the use of dummy electrodes in multiple working electrode systems as a means of lowering or Test strips for methods that eliminate the effects of interfering compounds may increase the cost and complexity of test strip production.

Using multiple working electrodes to measure an analyte, some test strip designs for systems such as those used in the OneTouch ^® Ultra ^® measurement system is 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 methods to reduce or eliminate the effect of interfering compounds without the use of dummy electrodes, intermediate films, or redox mediators with low redox potentials.

Invention Overview

The present invention relates to methods of reducing the effect of interfering compounds in the measurement of an analyte, and more particularly, to methods of reducing the effect of interfering compounds in systems in which test strips use two or more working electrodes. In one embodiment of the invention, a first potential is applied to the first working electrode and a second potential is applied to the second working electrode, said second potential having the same polarity as the first potential, but Its magnitude is larger than the first potential. For embodiments where reduction current is used to measure analyte concentration, the second potential may also be smaller in magnitude than the first potential. In one embodiment, the first working electrode and the second working electrode can be covered with an analyte-specific enzymatic reagent and a redox mediator. The first potential applied to the first working electrode is chosen such that it is sufficient to oxidize the reduced redox mediator in a diffusion-limited manner, while the second potential is chosen to be larger (i.e., in absolute value) than the first potential large, 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 includes the analyte current and the interfering compound current, and the current measured at the second working unit includes 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 the current corresponding to the concentration of the analyte and which is the result of oxidation of the reduced mediator. In one embodiment of the 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 formula,

${\mathrm{<span\; class="notranslate">\; A</span>}}_{\mathrm{<span\; class="notranslate">\; 1</span>}}<span\; class="notranslate">\; =</span>\frac{{\mathrm{<span\; class="notranslate">\; W</span>}}_{\mathrm{<span\; class="notranslate">\; 2</span>}}<span\; class="notranslate">\; -</span>{\mathrm{<span\; class="notranslate">\; YW</span>}}_{\mathrm{<span\; class="notranslate">\; 1</span>}}}{\mathrm{<span\; class="notranslate">\; x</span>}<span\; class="notranslate">\; -</span>\mathrm{<span\; class="notranslate">\; Y</span>}}$

where _A1 is the analyte current at the first working electrode, _W1 is the current measured at the first working electrode, _W2 is the current measured at the second working electrode, and X is the analyte-dependent voltage effect factor , and Y is the interfering compound-dependent voltage effect factor. Using the above formula in the method of the present invention can reduce the influence of the oxidation current caused by the presence of interfering compounds, and calculate a corrected current value that is more representative of the concentration of the analyte in the tested sample.

In one embodiment of the invention, the glucose concentration in a sample placed on a test strip can be calculated as follows: The sample is placed on a test strip having a first working electrode and a second working electrode. Electrodes and reference electrodes, at least the first working electrode and the second working electrode are covered with compounds (such as enzymes and redox mediators), when between the first working electrode and the reference electrode and the second working electrode The compound is adapted to promote the oxidation of glucose and the transfer of electrons from oxidized glucose to the first working electrode and the second working electrode when a potential is applied between the reference electrode. According to the present invention, a first potential is applied between the first working electrode and the reference electrode, the size of the first potential is selected so that it is sufficient to ensure that the magnitude of the current due to the oxidation of glucose in the sample is only affected by the applied Limitations by factors other than voltage, such as diffusion. According to the invention, a second potential is applied between the second working electrode and the reference electrode, the magnitude of the second potential is greater than the first potential, and in one embodiment of the invention, the second potential is selected to increase the oxidation of interfering compounds at the second working electrode. In another embodiment of the present invention, the following formula can 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 glucose concentration can be derived using the calculated current A _1G , where

${\mathrm{<span\; class="notranslate">\; A</span>}}_{\mathrm{<span\; class="notranslate">\; 1</span>}\mathrm{<span\; class="notranslate">\; G</span>}}<span\; class="notranslate">\; =</span>\frac{{\mathrm{<span\; class="notranslate">\; W</span>}}_{\mathrm{<span\; class="notranslate">\; 2</span>}}<span\; class="notranslate">\; -</span>{\mathrm{<span\; class="notranslate">\; YW</span>}}_{\mathrm{<span\; class="notranslate">\; 1</span>}}}{{\mathrm{<span\; class="notranslate">\; x</span>}}_{\mathrm{<span\; class="notranslate">\; G</span>}}<span\; class="notranslate">\; -</span>\mathrm{<span\; class="notranslate">\; Y</span>}}$

where _A1G is the glucose current, _W1 is the current measured at the first working electrode, _W2 is the current measured at the second working electrode, _XG is the glucose-dependent voltage effect factor, and Y is the interfering compound Dependent voltage effect factor.

Brief description of attached drawings

The novel features of the invention are given especially in the claims. A better understanding of the features and advantages of the present invention may be obtained from the following detailed description of exemplary embodiments, using the principles of the invention and the accompanying drawings:

Figure 1 is an exploded perspective view of an embodiment of a test strip useful in the present invention.

Figure 2 is a diagrammatic view of the meter and test strip used in the present invention.

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

                    Invention Details

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 this 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 enhance selectivity using the methods of the present invention include electrochemical sensors for measuring the concentrations 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 although the method of the present invention is described in the context of an electrochemical system in which the measured current is generated by oxidation, the invention is equally applicable to systems in which the measured current is generated by reduction.

The invention relates to a method of increasing the selectivity of an electrochemical measurement system, which method is particularly suitable for blood glucose measurement systems. More specifically, the present invention relates to a method of improving the selectivity of a blood glucose measurement system by partially or completely correcting for the effects of directly interfering 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 generate interfering currents. Thus, increased selectivity reduces the current generated at the working electrode due to the presence of interfering compounds (ie, compounds other than glucose that oxidize to produce interfering currents) 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 acetaminophen (Tylenol ^™ or Paracetamol), ascorbic acid, bilirubin, dopamine, gentisic acid, glutathione Glycine, Levodopa, Methyldopa, Tolazamide, Tolbutamide, and Uric Acid. Such interfering compounds can be oxidized, for example by chemical reaction with redox mediators, or by oxidation on the electrode surface.

In a fully selective system, there would be no oxidation current generated by any interfering compound, and the entire oxidation current would be generated by oxidation of glucose. However, if the oxidation of the interfering compound and the resulting oxidation current cannot be avoided, the present invention describes methods of eliminating some or all of the effects of the interfering compound by quantitatively determining the proportion of the oxidation current generated by the interfering compound to the total oxidation current , and this current is subtracted from the total oxidation current. In particular, in the method of the invention, glucose and interfering compounds are estimated using a test strip comprising a first working electrode and a second working electrode, applying two different potentials, and measuring the oxidation current generated at each working electrode. The respective proportions of the oxidation current.

In one embodiment of the method of the invention, the test strip used comprises a sample chamber comprising 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, glucose oxidase is reduced by glucose in the blood sample to produce 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 may be suitable for use in the methods of the invention is the ^{OneTouch® Ultra®} test strip sold by LifeScan, Inc. of Milpitas, California. Other test strips are described in International Publications WO 01/67099A1 and WO 01/73124A2.

In one embodiment of the method of the invention, a first potential is applied to the first working electrode and a second potential is applied to the second working electrode. In this embodiment, the magnitude of the first potential is chosen 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 determined by the reduced Limitation of the amount of redox mediators. It should be noted that glucose was not directly oxidized at the working electrode, but was indirectly oxidized by using redox enzymes and redox mediators. In the context of the present invention, glucose current refers to the oxidation of reduced redox mediators in relation to glucose concentration. In embodiments of the invention, when 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 about 385 millivolts to about 415 millivolts, even more preferably about 395-405 mV. Apply a second potential to the second working electrode such that the second potential is greater than the first potential. Wherein the applied potential is greater than that 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 mV to about 1000 mV, even more preferably from about 395-405 mV.

Since 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 at the second working electrode. The potential on the electrode is greater than the potential on the first working electrode. Therefore, any additional current measured at the second working electrode can be attributed to the oxidation of interfering compounds. In other words, the higher potential on the second working electrode should cause the glucose overpotential current measured on the second working electrode to be equal or substantially equal in magnitude to the glucose current on the first working electrode because The first sum potential and the second potential are in the restricted glucose current range, which is insensitive to changes in the applied potential. In practice, however, other parameters may affect the measured current, for example, when a higher potential is applied to the second working electrode, the total The current will often increase slightly. When there is an IR drop (ie uncompensated resistance) in the system, a higher applied potential causes an increase in the measured current. Examples of IR drop can be the first working electrode, the second working electrode, the reference electrode, the nominal resistance of 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 double layer that forms at the electrode/liquid interface, increasing the ionic capacitance at either the first working electrode or the second working electrode and the resulting current.

In order 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 the glucose overpotential current. The proportional relationship between the glucose current and the glucose overpotential current can be described by the following formula.

X _G ×A _1G ＝A _2G (Formula 1)

where X _G is the glucose-dependent voltage effect factor, A _1G is the glucose current at the first working electrode, and A _2G is 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 effect factor can be expected to be from 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 a diffusion-limited situation.

In the embodiments of the invention described above, glucose is measured indirectly by oxidation of ferrocyanide at the working electrode, and the concentration of ferrocyanide is directly proportional to the concentration of glucose. For a particular electrochemical compound, the standard potential (E°) value is a measure of the compound's ability to exchange electrons with other compounds. In the method of the invention, the potential at the first working electrode is chosen such that it is greater than the standard potential (E°) of the redox mediator. Because the first potential is chosen such that it is sufficiently larger than the E° value of the redox pair, the oxidation rate does not increase significantly with increasing applied potential. Therefore, 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 interfering compounds Oxidation.

Figure 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 and reference electrodes. Figure 3 shows that at about 400 mV, the current forms the onset of a plateau region because the applied voltage is sufficiently greater than the E° value of ferrocyanide. Therefore, as shown in Figure 3, the glucose current becomes saturated when the potential reaches about 400 mV, because the oxidation of ferrocyanide is diffusion-limited (diffusion of ferrocyanide to the working electrode limits the measured current size, and is not limited by the electron transfer rate between the ferrocyanide and the electrode).

In general, the current generated by the oxidation of interfering compounds does not saturate with an increase in the applied voltage, and compared to the current generated by the oxidation of ferrocyanide (ferrocyanide has passed through glucose and enzyme and enzyme and ferrous produced by the interaction of ferricyanide), showing a much stronger dependence on the applied potential. Interfering compounds generally have slower electron transfer kinetics than redox mediators (ie, ferrocyanides). The reason for this difference is the fact that most interfering compounds undergo the inner-sphere electron transfer route, whereas ferrocyanides undergo the faster outer-sphere electron transfer route. Typical inner-sphere electron transfer pathways require chemical reactions, such as hydride transfer, to occur before transferring electrons. In contrast, the exosphere electron transfer pathway does not require chemical reactions before transferring electrons. Therefore, inner-sphere electron transfers are generally slower than outer-sphere electron transfers because they require additional chemical reaction steps. Oxidation of ascorbic acid to dehydroascorbic acid is an example of endosphere oxidation, which requires the release of two hydride moieties. The oxidation of ferricyanide to ferrocyanide is an example of outer-sphere electron transfer. Therefore, the current generated by interfering compounds generally increases when tested at higher potentials.

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

Y×I ₁ =I ₂ (Formula 2)

where Y is the interfering compound-dependent voltage effect factor, _I1 is the interfering compound current, and _I2 is the interfering compound overpotential current. Because the interfering compound-dependent voltage effect factor Y depends on many factors, including the specific interfering compound and the material of the working electrode, for a specific system, test strip, analyte, and interfering compound, the specific interfering compound-dependent voltage effect factor The calculations may require experiments to optimize the voltage effect factors for these standards. Alternatively, under some conditions, an appropriate voltage effect factor can be derived or described mathematically.

In an 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 can be calculated using Tafel's formula for _I1 and _I2 mathematical means to describe,

$I_1=a^{\&prime;}\exp \left(\frac{{\mathrm{\&eta;}}_1}{b^{\&prime;}}\right)$ (Formula 2a)

$I_2=a^{\&prime;}\exp \left(\frac{{\mathrm{\&eta;}}_2}{b^{\&prime;}}\right)$ (Formula 2b)

where η ₁ =E ₁ -E°, η ₂ =E ₂ -E°, b' is a constant depending on the specific electroactivity interfering compound, E ₁ is the first potential, and E ₂ is the second potential. The value of E° (the standard potential of the particular interfering compound) is not important since it is canceled out in the calculation of Δη. Formulas 2, 2a, and 2b can be combined and rearranged to obtain the following formula,

$Y=\exp \left(\frac{\mathrm{\&Delta;\&eta;}}{b^{\&prime;}}\right)$ (Formula 2c)

where Δη=E ₁ -E ₂ . Equation 2c provides a mathematical relationship describing the relationship between Δη (ie, the difference between the first potential and the second potential) and the interfering compound-dependent voltage effect factor Y. In one embodiment of the present invention, Y may range from about 1 to about 100, more preferably from about 1-10. In one embodiment of the invention, for a particular interfering compound or combination of interfering compounds, the interfering compound-dependent voltage effect factor Y can be determined experimentally. It should be noted that for interfering compounds, the interfering compound-dependent voltage response factor Y is generally greater than the voltage response factor X _G for glucose. As described in the following sections, a) the mathematical relationship between the interfering compound current I ₁ and the interfering compound overpotential current I ₂ ; and b) the mathematical relationship between the glucose current A _1G and the glucose overpotential current A _2G enables to propose A glucose algorithm that reduces the effect of interfering compounds on glucose measurements.

In one embodiment of the invention, an algorithm was developed to calculate corrected glucose currents (ie A _1G and A _2G ) that are not affected by interferents. After the sample is applied 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, the first current is measured, which can be described by the following formula,

W ₁ =A _1G +I ₁ (Formula 3)

where _W1 is the first current at the first working electrode. In other words, the first current consists of the superposition of the glucose current A _1G and the interfering compound current I ₁ . More specifically, the interfering compound current may be the direct interfering current described above. At the second working electrode, measure the second current at the second potential or superpotential, which can be described by the following formula,

W ₂ =A _2G +I ₂ (Formula 4)

where _W2 is the second current at the second working electrode, _A2G is the glucose overpotential current measured at the second potential, and _I2 is the interfering compound overpotential current measured at the second potential. More specifically, the interfering compound overpotential current may be the direct interfering compound current described above. Using the 4 equations described above (Equations 1-4) including the 4 unknowns (A _1G , A _2G , I ₁ and I ₂ ), a corrected glucose current equation unaffected by interfering compounds can be calculated.

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

W ₂ =X _G A _1G +YI ₁ (Formula 5)

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

YW ₁ =YA _1G +TI ₁ (Formula 6)

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

W ₂ -YW ₁ =X _G A _1G -YA _1G (Formula 7)

Equation 7 is rearranged to solve for the corrected glucose current A _1G measured at the first potential, as shown in Equation 8.

$A_{1G}=\frac{W_2-{\mathrm{YW}}_1}{x_G-Y}$ (Formula 8)

The corrected glucose current A _1G is obtained by Equation 8, which eliminates the influence of interference, which only requires the output currents of the first working electrode and the second working electrode (such as W ₁ and W ₂ ), the glucose-dependent voltage effect factor Interfering compound-dependent voltage-effect factor Y for X _G and interfering compounds.

A glucose meter containing electronics is electrically connected to the glucose test strip to measure current from _W1 and _W2 . In one embodiment of the invention, _XG and Y can be programmed into the glucose meter so that only the memory is read. In another embodiment of the present invention, Y can be communicated to the meter through a calibration code chip. The calibration code chip has in its memory a specific set of values for _XG and Y that can be corrected for many specific test strips. This could explain the lot-to-lot variance of test strips that might have occurred in X _G and Y.

In another embodiment of the invention, the corrected glucose current in Equation 8 may be used by the meter only when some threshold is exceeded. For example, if _W2 is 10% or more greater than _W1 , the meter will use Equation 8 to correct the output current. However, if _W2 is 10% or less than _W1 , the concentration of interfering compounds is very low, so the measuring instrument can simply take the average current value between _W1 and _W2 to improve the measurement accuracy and Accuracy. Instead of simply averaging currents W ₁ and W ₂ , a more accurate approach could be to average W ₁ using W ₂ /X _G , which takes into account the glucose-dependent voltage effect factor X _G (note that when I ₂ is low , according to formulas 1 and 4, W ₂ /X _G is approximately equal to A _1G ). The strategy of using Equation 8 mitigates the risk of overcorrecting the measured glucose currents only in some cases where significant levels of interfering compounds are present in the sample. It should be noted that when _W2 is sufficiently greater than _W1 (eg, about 100% or more greater), this is indicative of a very high concentration of interfering compounds. In such cases, it may be desirable to output an error message instead of a glucose value, since very high levels of interfering compounds can cause the accuracy of Equation 8 to break.

The following sections 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 comprising six layers disposed on a substrate 5 . The six layers are conductive layer 50 , insulating layer 16 , reagent layer 22 , adhesive layer 60 , hydrophilic layer 70 and top layer 80 . The test strip 600 can be manufactured in a series of steps in which the conductive layer 50, the insulating layer 16, the reagent layer 22, the adhesive layer 60 are arranged on the substrate 5 using eg a screen printing method. Hydrophilic layer 70 and top layer 80 can be removed from roll stock and laminated to substrate 5. The fully assembled test strip forms a sample receiving chamber that receives a blood sample so that the blood sample can be analyzed.

The conductive layer 50 includes a reference electrode 10, a first working electrode 12, a second working electrode 14, a first contact 13, a second contact 15, a reference contact 11 and a 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 used for the conductive layer may be carbon ink such as those described in US5653918.

The insulating layer 16 includes a cutout 18 that exposes a portion of the reference electrode 10, the first working electrode 12, and the 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 Insulayer 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 invention, reagent layer 22 may include chemicals that selectively react with glucose, such as redox enzymes and redox mediators. During this reaction, a proportional amount of reduced redox mediator can be produced, which can be measured electrochemically, allowing the glucose concentration to 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, all of which are incorporated herein by reference.

Adhesive layer 60 includes first adhesive pad 24 , second adhesive pad 26 and third adhesive pad 28 . The side edges of the first adhesive pad 24 and the second adhesive pad 26 adjacent to the reagent layer 22 respectively define the walls 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 Tape Specialties LTD in Tring, Herts, United Kingdom (part &num; A6435).

The 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 polyester with a hydrophilic surface such as an anti-fog coating, which is commercially available from 3M. It should be noted that both the distal hydrophilic membrane 32 and the proximal hydrophilic membrane 34 are transparent to enable the user to observe the liquid sample filling the sample receiving chamber.

Top layer 80 includes transparent portion 36 and opaque portion 38 . The top layer 80 is disposed on and bonded to the hydrophilic layer 70 . As a non-limiting example, top layer 40 may be polyester. It should be noted that the transparent portion 36 substantially overlaps the proximal hydrophilic pad 32, which enables the user to visually verify that the sample receiving chamber is sufficiently filled. Opaque portion 38 assists the user in viewing the height contrast between the colored fluid, such as blood, within the sample receiving chamber and the opaque region of the apical membrane.

FIG. 2 is a simplified diagrammatic view showing meter 500 connected to test strip 600 . Meter 500 has three electrical contacts that form 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 testing, the voltage source 103 in the measuring instrument 500 applies a first potential _E1 between the first working electrode 12 and the reference electrode 10, and the voltage source 104 applies a voltage between the second working electrode 14 and the reference electrode 10. A second potential E ₂ is applied between them. The blood sample is applied such that the first working electrode 12, the second working electrode 14 and the reference electrode 10 are covered with blood. This causes the reagent layer 22 to hydrate, producing ferrocyanide in an amount proportional to the concentration of glucose and/or interfering compounds present in the sample. Five seconds after application of the sample, the meter 500 measures the oxidation currents of the first working electrode 12 and the second working electrode 14 .

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

It should be appreciated that for structures illustrated and described herein, equivalent structures may be substituted and that the described embodiments of the invention are not the only structures that may be used in the present invention. In addition, it should be understood that each structure described above has a function, and such a structure can 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 illustration only. Many changes, changes and substitutions will 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 included therein.

Claims (8)

1. A method of reducing interference in an electrochemical sensor, the method comprising: Apply the first potential to the first working electrode; applying a second potential to the second working electrode, wherein the second potential is greater than the absolute value of the 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 on the second working electrode, the second current comprising an analyte overpotential current and an interfering compound overpotential current, wherein the analyte overpotential current and the analyte current have a first proportional relationship, and wherein said interfering compound overpotential current has a second proportional relationship to said interfering compound current; and A corrected current value representative of the analyte concentration is calculated using a formula that is a function of the first current, the second current, the first proportional relationship, and the second proportional relationship. 2. The method of claim 1, wherein said formula is ${\mathrm{<span\; class="notranslate">\; A</span>}}_{\mathrm{<span\; class="notranslate">\; 1</span>}}<span\; class="notranslate">\; =</span>\frac{{\mathrm{<span\; class="notranslate">\; W</span>}}_{\mathrm{<span\; class="notranslate">\; 2</span>}}<span\; class="notranslate">\; -</span>{\mathrm{<span\; class="notranslate">\; YW</span>}}_{\mathrm{<span\; class="notranslate">\; 1</span>}}}{\mathrm{<span\; class="notranslate">\; x</span>}<span\; class="notranslate">\; -</span>\mathrm{<span\; class="notranslate">\; Y</span>}}$ where _A1 is the analyte current, _W1 is the first current, _W2 is the second current, X is the analyte voltage effect factor, and Y is the interfering compound voltage effect factor. 3. The method of claim 1, wherein the analyte is glucose. 4. The method of claim 1, wherein for said electrochemical sensor comprising a carbon working electrode and a ferrocyanide redox mediator, said first potential is from about 385 millivolts to about 415 millivolts. 5. The method of claim 1, wherein said second potential is from about 420 millivolts to about 1000 millivolts for said electrochemical sensor comprising a carbon working electrode and a ferrocyanide redox mediator. 6. The method of claim 1, wherein said interfering compound current is generated due to the oxidation of at least one compound selected from the group consisting of paracetamol, ascorbic acid, bilirubin, dopamine, gentisic acid, glutathione, L- Dopa, methyldopa, tolazamide, tolbutamide, and uric acid. 7. The method of claim 1, wherein said first proportional relationship is XxA ₁ =A ₂ where X is the analyte voltage effect factor, _A1 is the analyte current, and _A2 is the analyte overpotential current. 8. The method of claim 1, wherein said second proportional relationship is YxI ₁ =I ₂ where Y is the interfering compound voltage effect factor, _I is the interfering compound current, and _I is the interfering compound overpotential current.

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