MXPA06008843A - Oxidizable species as an internal reference for biosensors and method of use - Google Patents

Abstract

A biosensor (102) for determining the presence or amount of a substance in a sample and methods of use of the biosensor (102) are provided. The biosensor (102) for receiving a user sample to be analyzed includes a mixture for electrochemical reaction with an analyte. The mixture includes an enzyme, a mediator and an oxidizable species as an internal reference.

Description

OXIDABLE SPECIES AS INTERNAL REFERENCE FOR BIOSENSORS AND METHOD OF USE FIELD OF THE INVENTION The present invention relates generally to a biosensor and, more particularly, to a new and improved biosensor, which includes an oxidizable species as an internal reference and methods of using the biosensor, to determine the presence or amount of a substance in a sample.

DESCRIPTION OF THE PREVIOUS TECHNIQUE The quantitative determination of analytes in body fluids is of great importance in the diagnosis and maintenance of certain physiological abnormalities. For example, lactate, cholesterol and bilirubin should be monitored in certain individuals. In particular, the determination of glucose in body fluids is of great importance for diabetic individuals who must frequently check the glucose level in their body fluids as a means to regulate the glucose intake in their diets. While the rest of the description of this document will be directed towards the determination of glucose, it should be understood that the new and improved sensor element and the method of use of this invention can be used for the determination of other analytes with the selection of the enzyme appropriate The methods for determining the concentration of analytes in fluids can be based on the electrochemical reaction between the analyte and a specific enzyme for the analyte and a mediator that maintains the enzyme in its initial oxidation state. Suitable redox enzymes include oxidases, dehydrogenases, catalase and peroxidase. For example, in the case where glucose is the analyte, the reaction with glucose oxidase and oxygen is represented by the equation: Glucose oxidase (GO) Glucose + 02 - > gluconolactone + H202 (A) In the initial step of the reaction represented by equation (A), the glucose present in the test sample converts the enzyme (Eox), such as the oxidized center of flavin-adenine-dinucleotide (FAD) of the enzyme into its form reduced (Ered), for example, (FADH2). Because these redox centers are electrically isolated essentially within the enzyme molecule, the direct transfer of electrons to the surface of a conventional electrode does not occur to any measurable degree in the absence of an unacceptably high cell voltage. An improvement to this system involves the use of a non-physiological redox coupling between the electrode and the enzyme to alternate electrons between the (FADH2) and the electrode. This is represented by the following scheme in which the redox coupler, typically referred to as a mediator, is represented by M: Glucose + GO (FAD) -? - gluconolactone + GO (FADH2) GO (FADH2) + 2Mox? GO (FAD) + 2Mre + 2H + 2Mred - 2Mox + 2e "(on the electrode) In the scheme, GO (FAD) represents the oxidized form of glucose oxidase and G0 (FADH2) indicates its reduced form. The mediating species M0? / Mred alternates electrons from the reduced enzyme to the oxidizing electrode with which the enzyme causes its regeneration in situ. U.S. Patent Nos. 5,620,579 and ,653,863 issued to Gensha et al., And assigned to the present assignee, describe apparatuses and a method for determining the concentration of an analyte in a test sample of the fluid by applying the test sample of the fluid to the surface of a working electrode, the which is electrochemically connected to a counter electrode and whose surface carries a composition comprising an enzyme specific for the analyte. A mediator is reduced in its response to a reaction between the analyte and the enzyme. An oxidation potential is applied between the electrodes to return at least a portion of the mediator back to its oxidized form before determining the concentration of the analyte to increase thereby the accuracy of the analyte determination. After this initially applied potential, the circuit is switched to an open circuit or to a potential that substantially reduces the current to minimize the proportion of the electrochemical potential at the working electrode. A second potential is applied between the electrodes and the current generated in the test sample of the fluid is measured to determine the concentration of the analyte. Optionally, the accuracy of the analyte determination is further improved algorithmically.

BRIEF DESCRIPTION OF THE INVENTION Important aspects of the present invention are to provide a new and improved biosensor for determining the presence or amount of a substance in a sample that includes an oxidizable species as an internal reference and a method of using the biosensor. In summary, a biosensor is provided to determine the presence or quantity of a substance in a sample and the methods of using the biosensor. The biosensor for receiving a sample from the user to be analyzed includes a mixture for an electrochemical reaction with an analyte. The mixture includes an enzyme, a mediator and an oxidizable species as internal reference. The internal reference is defined as the oxidizable species which in one embodiment can be further defined as the reduced form of a reversible redox coupling having a redox potential equal to or higher than that of the mediator. The internal reference acts to increase the response current by addition for the operation potentials that oxidize both species and in the case where glucose is the analyte, a total response current is represented by: Itotal = -E ± nt-ref + Iglucose I ± nt-ret ° c (internal reference) and Igiucos 8 (glucose); Where Iint-ref is the portion of the total response current due to the internal reference, whereas Ig? Ucosa is due to the oxidation of the mediator proportional to the glucose concentration. According to the characteristics of the invention, the internal reference can be either the same mediator species or an oxidizable species with a higher redox potential than the mediator. In this way, for biosensors with a low operating potential that oxidize only the mediator, the Iint-ref current will be zero. However, for biosensors with a higher operating potential that oxidizes both species, the total response current will be the sum of the portion due to internal reference and due to glucose. Since the concentration of the internal reference is fixed, the calibration slope of the sensor will depend solely on the response of the sensor for glucose while the intercept will depend on the aggregate amount of the internal reference. In other words, the internal reference will only divert the intercept and will not change the calibration slope. In this way, the concept of internal reference provides new and different ways to make glucose biosensors.

BRIEF DESCRIPTION OF THE DRAWINGS The present invention together with the above objects and advantages and still others can be better understood from the following detailed description of the preferred embodiments of the invention illustrated in the drawings, wherein: Figure IA is a representation of a block diagram of the biosensor meter including a biosensor having an internal reference according to the present invention; Figures IB, 1C and ID are diagrams respectively illustrating the methods of operation for use with the biosensor of Figure 1 of the invention; Figures 2A, 2B and 2C are diagrams showing three cyclic tensomograms of glucose biosensors based on MLB with ferrocyanide as the internal reference of the biosensor of Figure 1 of the invention in whole blood samples of 0 mg / dL of glucose; Figure 3 is a diagram illustrating a linear response of the biosensor of Figure 1 of the invention to different voltage operating potentials; Figure 4 is a diagram illustrating the effect of the internal reference added to the total tensometric current using the biosensors of Figure 1 of the invention with 10% ferricyanide printed as the counter electrode; Figures 5A and 5B are diagrams illustrating the linear response and the increased intercept with the increase of the internal reference of the MLB-based biosensors of Figure 1 of the invention with Ag / AgCl as the counter electrode; Figures 6A and 6B are diagrams illustrating the linear response and the increased intercept with the increase of the internal reference of MLB-based biosensors. Figure 1 of the invention with 10% ferricyanide as the counter electrode; Figure 7 is a diagram illustrating the linear relationship of the calibration intercept with the increase of the internal, increasing reference of the DEX biosensors of Figure 1 of the invention with 10% ferricyanide as the counter electrode; and Figures 8A and 8B are diagrams illustrating the signal relationship with respect to the reference results of the flow injection analysis (FIA) of the residual ferrocyanide of a reactive control ink and the reactive ink. with 0.1% ferrocyanide added to the 20% ferricyanide reactive mixture of a biosensor of Figure 1 of the invention.

DETAILED DESCRIPTION OF THE PREFERRED MODALITIES The present invention relates to an electrochemical biosensor for determining the presence or quantity of a substance in a sample. The biosensor includes sensor strips containing a working electrode and a counter electrode, each of which is at least partially covered with, for example, a separate layer of reagent. The reagent layer on the working electrode includes, for example, an enzyme that interacts with an analyte through an oxidation-reduction reaction and also includes a mediator which is the oxidized form of a redox coupling. The biosensor of the invention includes an internal reference or a reduced form of the mediator in the reagent layer on the working electrode. The internal reference is defined as an oxidizable species which in one embodiment can be further defined as a reduced form of a redox, reversible coupling having a redox potential equal to or higher than that of the mediator. A quantitative, fixed amount of the internal reference is provided in the reagent layer. The biosensors of the invention that include the internal reference or an aggregate amount of the reduced form of the mediator provide improvements in that the internal reference acts to ensure the interception of inherent thermodynamic calibration while maintaining the calibration slope. Many compounds are useful as mediators due to their ability to accept electrons from the reduced enzyme and transfer them to the electrode. A necessary attribute of a mediator is the ability to remain in the oxidized state under the conditions present on the surface of the electrode before the use of the sensor. Among the most venerable mediators are the oxidized form of organometallic compounds, organic molecules, transition metal coordination complexes. A specific example of a mediator is potassium hexacyanoferrate (III), also known as ferricyanide. As used in the following specification and claims, the term "biosensor" means a sensor strip, electrochemical or sensing element of an analytical device or an instrument that selectively responds to analytes in an appropriate sample and converts its concentration to an electrical signal. The biosensor directly generates an electrical signal, facilitating a simple instrument design. Also, a biosensor offers the advantage of low cost of the material since a thin layer of chemicals is deposited on the electrodes and little material is wasted. The term "sample" is defined as a composition that contains an unknown quantity of the analyte of interest. Typically, a sample for the electrochemical analysis is in liquid form and preferably the sample is an aqueous mixture. A sample may be a biological sample, such as blood, urine or saliva. A sample may be a derivative of a biological sample, such as an extract, a dilution, a filtrate or a reconstituted precipitate. The term "analyte" is defined as a substance in a sample, the presence or quantity of which must be determined. An analyte interacts with the oxidoreductase enzyme that is present during the analysis and can be a substrate for oxidoreductase, a coenzyme or another substance that affects the interaction between oxidoreductase and its substrate. The term "oxidoreductase" is defined as any enzyme that facilitates the oxidation or reduction of a substrate. The term oxidoreductase includes "oxidases", which facilitate oxidation reactions in which molecular oxygen is the electron receptor; "reductases", which facilitate the reduction reactions in which the analyte is reduced and the molecular oxygen is not the analyte; and "dehydrogenases", which facilitate oxidation reactions in which molecular oxygen is not the electron receptor. See, for example, Oxford Dictionary of Biochemistry and Molecular Biology, Revised Edi tion, A.D. Smith, Ed., New York: Oxford University Press (1997) pages 161, 476, 477 and 560. The term "oxidation-reduction" reaction is defined as a chemical reaction between two species that involves the transfer of at least one electron from one species to the other species. This type of reaction is also referred to as a "redox reaction". The oxidation portion of the reaction involves the loss of at least one electron by one of the species and the reduction portion involves the addition of at least one electron to the other species. The ionic charge of a species that is oxidized becomes more positive by an amount equal to the number of electrons transferred. Likewise, the ionic charge of a species that is reduced becomes less positive by an amount equal to the number of electrons transferred. The term "oxidation number" is defined as the ionic, formal charge of a chemical species, such as an atom. A higher oxidation number, such as (III), is more positive and a lower oxidation number, such as (II), is less positive. A neutral species has an ionic charge of zero. The oxidation of a species results in an increase in the oxidation number of that species and the reduction of one species results in a decrease in the oxidation number of that species. The term "redox couple" is defined as two species of a chemical substance that have different oxidation numbers. The reduction of the species that has the highest reduction number produces the species that has the lowest oxidation number. Alternatively, oxidation of the species having the lowest oxidation number produces the species having the highest oxidation number. The term "oxidizable species" is defined as the species of a redox pair that has the lowest oxidation number and which is thus capable of being oxidized to be the species with the highest oxidation number. Likewise, the term "reducible species" is defined as the species of a redox pair that has the highest oxidation number and which is thus able to be reduced to be the species that has the oxidation number. lower. The term "organotransition metal complex" also known as "OTM complex" is defined as a complex where a transition metal is linked to at least one carbon atom through a similar bond (formal charge of -1 at the carbon atom). carbon sigma bonded to the transition metal) or a pi bond (formal charge of 0 on the carbon atoms pi linked to the transition metal). For example, ferrocene is an OTM complex with two cyclopentadienyl rings (Cp), each linked through its five carbon atoms to an iron center by two pi bonds and a sigma bond. Another example of an OTM complex is ferricyanide (III) and its reduced ferrocyanide (II) counterpart, where six cyano ligands (formal charge of -1 in each of the 6 ligands) are linked through a sigma link to a center of iron through the carbon atoms of the cyano groups. The term "coordination complex" is defined as a complex having a well-defined coordination geometry, such as octahedral or square planar geometry. Unlike the OTM complexes, which are defined by their link, the coordination complexes are defined by their geometry. In this manner, the coordination complexes can be OTM complexes (such as the aforementioned ferricyanide) or complexes where non-metallic carbon atoms, such as heteroatoms including nitrogen, sulfur, oxygen and phosphorus, are linked to the center of the transition metal. For example, ruthenium hexaamine, or hexaaminorrutenate (11) / (III), is a coordination complex that has a well-defined octahedral geometry where six NH3 ligands (formal charge of 0 in each of the 6 ligands) are linked datively to the ruthenium center. Ferricyanide is also an example of the coordination complex that octahedral geometry has. A more complete description of organotransition metal complexes, coordination complexes, and transition metal linkages can be found in Coliman et al., Principies and Applications of Organ Translation Metal Chemistry (1987) and Miessler & Tarr, Inorganic Chemistry (1991). The term "mediator" is defined as a substance that can be oxidized or reduced and that can transfer one or more electrons between a first substance and a second substance. A mediator is a reagent in an electrochemical analysis and is not the analyte of interest. In a simplistic system, the mediator is subjected to a redox reaction with the oxidoreductase after the oxidoreductase has been reduced or oxidized through its contact with an appropriate substrate. This oxidized or reduced mediator is then subjected to the opposite reaction at the electrode and is regenerated at its original oxidation number. The term "organic, electroactive molecule" is defined as an organic molecule that does not contain a metal and that is capable of undergoing an oxidation or reduction reaction. Organic, electroactive molecules can behave as redox species and as mediators. Examples of organic, electroactive molecules include the coenzyme pyrroloquinoline-quinone (PQQ), benzoquinones and naphthoquinones, N-oxides, nitroso compounds, hydroxylamines, oxines, flavins, phenazines, phenothiazines, indophenols and indamines. The term "electrode" is defined as an electrically conductive substance that remains stationary during an electrochemical analysis. Examples of electrode materials include solid metals, metal pastes, conductive carbon, conductive carbon pastes and conductive polymers.

Referring now to the drawings, Figure 1 illustrates a biosensor meter designated as a set by the reference character 100 of the preferred embodiment and ordered according to the principles of the present invention. The biosensor meter 100 includes a biosensor 102 arranged in accordance with the principles of the present invention. The biosensor meter 100 includes a microprocessor 104 together with an associated memory 106 for storing a program and user data. The digital data of the microprocessor 104 is applied to a digital to analog (D / A) converter 108. The D / A converter 108 converts the digital data to an analogous signal. An amplifier 110 coupled to the D / A converter 108 amplifies the analog signal. The amplified analog signal output of the amplifier 110 is applied to the biosensor 102 of the invention. The biosensor 102 is coupled to an amplifier 112. The signal from the amplified sensor is applied to an analog-to-digital (A / D) converter 114 that converts the analog sensor signal, amplified to a digital signal. The digital signal is applied to the microprocessor 104. Most disposable, commercially available biosensors that are used to monitor blood glucose require the deposition / printing of a mixture of an enzyme and a mediator with some binding agent. For the application of glucose measurement, the mediator is in the oxidized form of a redox coupling. Depending on the redox coupling, the mediator can be a very strong oxidant, such as ferricyanide, chemically oxidizing whereby the functional groups after mixing with the enzyme and the binding agent. Subsequently, a small amount of the reduced mediator is formed as an impurity in the reagent in the mixing, storage and printing processes. In this way, the final result of the mixing and printing of the reactive ink is the generation of the reduced form of the redox coupling, giving rise to the background current. The formation of this reduced form of the mediator and thus the background current may vary from batch to batch. This reduced form of the mediator generated by the process, such as ferricyanide ferrocyanide, can be oxidized in general to minimize the background signal using the algorithm outlined in U.S. Patent Nos. 5,620,579 and 5,653,863 issued to Genshaw et al., And assigned herein. assignee. However, the process-dependent background signal, which is translated into the calibration intercept, can be extended over a range of values. At the extremes of these values diverged from the intercept, an analytical precision will be experienced because a reasonable calibration intercept can not be assigned to accommodate the diverged intercept. In accordance with the features of the invention, a degree of mediator containing a certain level of the reduced form of the mediator in the reagent is used to decrease the effect of the strong oxidant. Thermodynamically, the presence of a small amount of the reduced form of the mediator in the enzyme and mediator ink mixture decreases the driving force for the conversion of the oxidized form to the reduced form. This is advantageously done by adding a small fixed amount of the reduced form of the mediator to the oxidized mediator. Although a background signal will be generated, the algorithm in U.S. Patent Nos. 5,620,579 and 5,653,863 will minimize the effect of the background to increase the accuracy of the glucose sensor. The patents identified above describe a method that reduces background polarization due to oxidizable impurities in an amperometric sensor used to measure a specific analyte, such as blood glucose. The background current of this sensor will increase if it is stored for a prolonged period of time or under tension (heat, humidity, etc.) due to the increased presence of the reduced mediator or other reduced impurity that is present in the sensor such as enzymatic stabilizers, for example glutamate, and surfactants that have reduction equivalents. For example, in an amperometric sensor based on ferricyanide, the background polarization is related to the presence of ferrocyanide (from the ferricyanide reduction) near the surface of the electrode. This accumulated ferrocyanide, which is opposite to the ferrocyanide produced during the use of the sensor (fresh ferrocyanide), is oxidized again to ferricyanide to reduce the background polarization that causes and extends thereby the useful life of the sensor. To achieve this goal, the method uses an electrochemical approach. The background polarization is further reduced when the electrochemical approach is enhanced with an algorithmic correction. The described method involves first applying a positive potential impulse (called the "reburn" impulse) which precedes the normal potential profile during the use of the biosensor. This is typically done by applying a positive potential from 0.1 to 0. 9 volts (preferably 0.3 to 0.7 volts) between the working electrode and the reference electrode of the sensor for a period of 1 to 15 seconds (preferably 5 to 10 seconds). The bursting impulse oxidizes the initial ferrocyanide (or other oxidizable impurity), so that the sensor can start the test with a clean bottom. Typically, the bottom is not perfectly clean since only a portion of the oxidizable impurity is oxidized by the bursting impulse. This is the case because the chemical layer covers both the working electrode and the counter electrode. The initial ferrocyanide exists in the chemical layer since it comes from ferricyanide. When the sample fluid is applied and the chemical layer is rehydrated, the ferrocyanide near the working electrode is re-oxidized. The rest of the ferrocyanide diffuses into the sample fluid and mixes with the glucose. That portion of the initial ferrocyanide can not be re-oxidized without affecting the glucose. The initial ferrocyanide is near the electrode for a very short time (a few seconds) after the test sample of the fluid is applied. The reason for this is that the chemicals (enzyme and ferricyanide, etc.) are deposited as a thin layer on the working electrode and the counter electrode. The reburning technique takes advantage of this since a significant amount of the initial ferrocyanide can be quenched without a noticeable reduction in the concentration of the analyte in the fluid test sample, most of which does not come into direct contact with the electrode . Experiments have shown that the background polarization of a sensor under tension can be reduced by 40% with the proper application of the reburn pulse.

The method described in U.S. Patent Nos. 5,620,579 and 5,653,863 is advantageously applied to minimize the effect of the background signal to increase the accuracy of the glucose biosensor meter 100 of the preferred embodiment. The subject matter of the patents identified above is incorporated herein by way of reference. In accordance with the features of the invention, the aggregate amount of the reduced form of the mediator acts to ensure the interception of inherent thermodynamic calibration while maintaining the calibration slope. In view of the function that the reduced form of the mediator, for example ferrocyanide, plays in the glucose sensor, it is preferred as the internal reference. Examples of mediators of electroactive organic molecules are described in U.S. Patent No. 5,520,786, issued to Bloczynski et al. On May 28, 1996 and assigned to the present assignee. In particular, a disclosed mediator (compound 18 in TABLE 1) coming 3-phenylimino-3H-phenothiazine referred to herein as MLB-92, has been used to make a glucose biosensor 102 according to the features of the invention . The subject matter of the patent identified above is incorporated herein by way of reference. A commercially available biosensor meter and biosensor are manufactured and sold by Bayer Corporation under the trademark Ascensia DEX. The Ascensia DEX biosensor generally includes a form of ferricyanide as pure as possible for the reagent. The Ascensia DEX biosensor has been used to make a glucose biosensor 102 according to the characteristics of the invention by adding an adequate amount of ferrocyanide to the pure ferricyanide. The benefits of the ferrocyanide addition that defines the internal reference of the biosensor 102 to the Ascensia DEX reactive ink include an immediate benefit in the increase of the intercept without changing the slope, ensuring the intercept interval and increasing the long-term stability of the biosensor. during storage. In accordance with the features of the invention, the MLB-92 mediator having a lower redox potential was used to make a glucose biosensor 102 with special properties. With the addition of adequate amounts of the internal reference, ferrocyanide, the new biosensor system can be made to work with two operation potentials; (1) at 400 mV where both the new mediator and the internal reference are oxidized and (2) at 100 mV where only the new mediator can be oxidized. The significance of this approach is twofold. First, the glucose biosensor 102 as formulated (new mediator and internal reference) can be operated at a high potential (+400 mV) to produce currents in a range that conforms to the calibration characteristics of the physical equipment requirements of the existing instrument. Second, since it is likely that the lower redox potential and thus a lower oxidation potency of the mediator will virtually not have a conversion of the oxidized form to the reduced mediator form, a lower operation potential can be applied (0). - 100 mV) to the sensor to avoid oxidation of the internal reference. In this way, a new set of calibration characteristics based on the new mediator, most likely with an almost zero intercept due to the lower oxidation power, will lead to better analytical accuracy for glucose measurements. It will also reduce the matrix interference in whole blood by preventing the oxidation of any of the oxidizable species, known as uric acid and acetaminophen. In accordance with the features of the invention, another application of the internal reference for glucose sensors 102 is to add a suitably large amount of the internal reference to the biosensor system to produce a high current response. Using the algorithm with double number of steps with an open circuit between them (Bayer patents # 5, 620,579 and # 5,653,863), the first potential step is set at 400 V to produce a current that is mainly due to the internal reference signal while the second step is set at a low potential (0 - 100 mV) to produce a current signal related to glucose concentration only. The ratio of the first signal, which must be virtually independent of the hematocrit of whole blood, with respect to the second signal at a low potential can be used to correct the analytical polarization due to the effect of the hematocrit. According to the characteristics of the invention, the internal reference is defined as the oxidizable species which in one embodiment is further defined as the reduced form of a redox, reversible coupling having a redox potential equal to or higher than that of the mediator . The concept and use of an internal reference are very common in the field of analytical chemistry. However, an example has not been suggested to use an internal reference for biosensors in the existing patents or bibliography. In the three scenarios described above, the internal reference acts to increase the response current by addition for the operating potentials that oxidize both species and with glucose as the analyte; A total response current is represented by: Itotal = -tint-ref "1" glucose int-ref ° c (internal reference) and Ig? UCose 8 (glucose); Where I ± nt-ref is I portion of the total response current due to the internal reference, while Igiucos is due to oxidation of the mediator proportional to the glucose concentration. According to the characteristics of the invention, the internal reference can be either the same mediator species or an oxidizable species with a higher redox potential than the mediator. In this way, for biosensors with a low operating potential that oxidizes only the mediator, the int-ref current will be zero. However, for biosensors with a higher operating potential that oxidizes both species, the total response current will be the sum of the portion due to internal reference and that due to glucose. Since the internal reference concentration is fixed, the calibration slope of the sensor will depend solely on the response of the sensor for glucose while the intercept will depend on the aggregate amount of the internal reference. In other words, the internal reference will only divert the intercept and will not change the calibration slope. In this way, the concept of internal reference provides new and different ways to make glucose biosensors. Referring now to Figures IB, 1C and ID, there are at least three modes of operation based on the use of an internal reference for the glucose biosensors 102 of the invention. Potentiostatically, the three modes of operation are represented in Figures IB, 1C and ID. Each of the illustrated modes of operation includes a first burn pulse, followed by a second standby or open circuit and a third final read pulse, each pulse or period having a selected duration, for example 10 seconds. In the basic or more intermediate operation, the ferrocyanide is retained in the ferricyanide at the concentration of 0.1 to 1% of the total ferricyanide providing the internal reference for the glucose biosensors 102 of the invention. This is represented in Figure IB where both potentials in the first and third periods are at the same voltage, for example 400 mV. The retention of a small percentage of ferrocyanide that defines the internal reference can be done either by means of an appropriate purification process of ferricyanide or by the addition of an adequate amount of ferrocyanide to the pure ferricyanide. The result of these retention processes is to deliberately maintain a desirable amount of ferricyanide in ferricyanide as a special grade of ferricyanide. This contrasts with the popular wisdom of having a form of ferricyanide as pure as possible, such as for the DEX reagent, usually ferrocyanide in the order of 0.05% ferricyanide or less as impurity. The most desirable amount is 0.1% ferrocyanide in the final formulation for the DEX sensor, which will lead to the assurance of the calibration intercept at a narrower range while maintaining the calibration slope for the DEX sensor. The second mode of operation is shown in Figure 1C, where a desirable amount of ferrocyanide (the internal reference) is added to the reagent of the enzyme and a mediator with a redox potential lower than that of the internal reference. It is expected that the biosensor 102 works under high and low potentials (for example at 400 mV and 100 mV against Ag / AgCl) for the existing instruments and instruments with a new requirement of the physical equipment. This biosensor can be operated in potential programs represented in Figure IB for the existing instruments 100 and Figure 1C for the new instruments 100. Examples of the combination of the mediator and the internal reference include the MLB-92 system and ferrocyanide as well as also ruthenium hexaamine and ferrocyanide. The separation of the two redox potentials is sufficiently large so that there will not usually be an oxidation of the internal reference species when operating at low voltage. The third mode of operation is shown in Figure ID, where a higher but desirable concentration of ferrocyanide is added to the reagent mixture of the enzyme and a mediator with a lower redox potential than that of the internal reference. The amount of the internal reference will preferably produce an equivalent current of about 50% to 75% of the full scale in the calibration range. In the operation algorithm, the first potential step is established to oxidize both the mediator and the internal reference (400 mV) while the second potential step for the reading impulse is to oxidize the mediator only (0 - 100 mV) . The current in the first potential step of Figure ID will be more relevant for the internal reference than immediately after for the electrode and should have virtually no effect on the hematocrit. The ratio of the current of the second step to that of the first step will provide a correction for the analytical polarization due to the effect of the hematocrit. Experiments have been carried out to show the feasibility of the method which consists of adding an internal reference to a mediating system to overcome the existing problems or to improve the performance of the sensor according to the biosensor 102 of the invention. Referring now to Figures 2A, 2B and 2C, three cyclic tomograms illustrating the operation of the biosensor 102 of the invention are shown. The three cyclic tomograms illustrated are for MLB 102-based glucose biosensors with ferrocyanide as the internal reference in whole blood samples of 0 mg / dL glucose. Figure 2A illustrates the working electrode against the ferricyanide counter electrode, Figure 2B illustrates the working electrode against the silver counterpart (Ag) and silver chloride (AgCl) or Ag / AgCl and the Figure 2C illustrates the working electrode against the counter electrode of MLB-92. The respective peaks labeled 1 and 2 represent the oxidation of the MLBred mediator (reduced form of MLB) and the internal reference of ferrocyanide respectively for the three tensomogram schemes. The oxidation peak for MLBred travels along the potential scale as the redox coupling in the counter electrode changes from ferricyanide to Ag / AgCl to MLB-92. However, it can be seen that the relative position of the MLB-92 mediator with respect to the internal ferrocyanide reference is the same in the three tensomogram schemes of Figures 2A, 2B and 2C. With reference to Figure 3, there is shown a diagram illustrating a linear response of the biosensor 102 of the invention at different voltage operating potentials. The biosensor 102 is operated at (1) a potential of 400 mV and (2) a potential of 150 mV. Figure 3 illustrates the response to the linear dose of the biosensor based on the MLB-92 mediator 102 with 20 mM ferrocyanide as the internal reference. The respective lines labeled as EXAMPLE 1 and EXAMPLE 2 are of operating potentials of 400 mV and 150 mV against the counter electrode of Ag / AgCl. As shown in Figure 3, biosensor 102 gives virtually the same slope but with different intercepts for potential operations of 400 mV and 150 mV. This result shows that the internal reference can be oxidized or selectively avoided by the operation potential. In this way, a biosensor 102 can serve two different meters. Examples of biosensor 102 have been systematically prepared by showing the increase in intercept with the increase in ferrocyanide as the internal reference while the slopes remained virtually unchanged. Three working electrode reagents were prepared in the following formulations. These three reagents were deposited with pins on two sensor formats: (1) Ag / AgCl as the counter electrode, (2) ferricyanide printed at 10% as the counter electrode.

Figure 4 illustrates the effect of the internal reference added to the total tensometric current using the biosensors 102 of the invention with 10% ferricyanide printed as the counter electrode. Figure 4 provides cyclic tensomograms of ferrocyanide sensors as the internal reference in whole blood samples of 0 mg / L glucose. The tensomograms labeled A, B and C are with the formulations 1, 2 and 3 respectively in their entirety with a counter-electrode of ferricyanide printed at 10%. The effect of the internal reference, added to the total tensometric current, is shown in Figure 4 using sensors with 10% ferricyanide printed as the counter electrode. The main oxidation / reduction peaks at this point are centered around -0.38 Volts against 10% ferricyanide, which is due to the MLB mediator. The oxidation peak at approximately 0 - 50 mV is due to the internal reference of the ferrocyanide. While the oxidation peak for the internal reference ferrocyanide increases with increases in the internal reference concentration from 0 to 4 to 8 mM, the oxidation peak for the mediator is virtually unchanged. At this point, the concept of internal reference is further explained by the fact that the MLBred main oxidation peak is unchanged by the presence of the internal reference. With reference to Figures 5A and 5B, diagrams illustrating the linear response and the increased intercept are shown with the increase of the internal reference of MLB-based biosensors 102 of the invention with Ag / AgCl as the counter electrode. Figure 5A illustrates the response to the linear dose of the biosensors based on MLB 102 with ferrocyanide 0, 4 and 8 mM, labeled respectively as EXAMPLE 1, EXAMPLE 2 and EXAMPLE 3. Figure 5B illustrates the intercept and slope as a function of the ferrocyanide added in the working electrode reagent of the biosensor 102 of the invention. The three sensors used Ag / AgCl as the counter electrode. Also with reference to Figures 6A and 6B, diagrams illustrating the linear response and the increased intercept are shown with the increase of the internal reference of the biosensors based on MLB 102 of the invention with 10% ferricyanide as the counter electrode. Figure 6A illustrates the linear dose response of biosensors based on MLB 102 with 0, 4 and 8 mM ferrocyanide labeled respectively as EXAMPLE 1, EXAMPLE 2 and EXAMPLE 3. Figure 6B illustrates the intercept and slope as a function of the ferrocyanide added to the working electrode reagent of the biosensor 102 of the invention. The three sensors used 10% ferricyanide printed as the counter electrode. In the dose response experiments, both series of Ag / AgCl counter-electrode sensors of Figures 5A and 5B and the 10% ferricyanide counter electrode of Figures 6A and 6B show a linear response and an increased intercept with the increase of the internal reference. For practical purposes, the slope of the three sensors in Figures 5A and 5B is unchanged while the intercept linearly increases with the added ferrocyanide. The same linear relationship of the intercept with added ferrocyanide and the flat slope trend are repeated in the series of sensors with% ferricyanide printed as the counter electrode, as shown in Figures 6A and 6B. Experiments have been carried out to show the addition of ferrocyanide to the DEX reactive ink, the modification of the calibration intercept without changing the slope according to the biosensor 102 of the invention. Figure 7 illustrates the linear relationship of the calibration intercept with the increase of the internal reference of the DEX-type biosensors 102 of the invention. Five different formulations in an established format labeled BC7 in Figure 7 were made with 0, 0.02, 0.04, 0.06 and 0.08% ferrocyanide mixed in the standard DEX reagent for the DEX sensor. The regression slope and intercepts for these five sensors of the BC7 format are shown in Figure 7. Except for the sensor with 0.06% ferrocyanide due to experimental problems, the intercepts of the other four sensors provide a nice linear function with respect to the added amount of ferrocyanide as the internal reference. On the other hand, the slopes of the five sensors are on a flat line indicating that the addition of the internal reference does not change the slope of the DEX 102 biosensors of the invention. Figures 8A and 8B illustrate the relationship of the signal to the reference results of the flux injection analysis (FIA) of the residual ferrocyanide of a control reactive ink and the reactive ink with 0.1% ferrocyanide added to the reagent mixture of 20% ferricyanide of a biosensor 102 of the invention. One of the subtle effects of the addition of the internal ferrocyanide reference to the DEX reactive ink is to decrease the driving force for the conversion of the ferricyanide mediator to ferrocyanide. In this way, the ferricyanide becomes the source of the residual current in the DEX sensor. One way to show this subtle effect is to monitor the increase in residual current (background current) of the reactive ink with the internal reference together with the control reactive ink for a prolonged period of time. Both reactive inks were stored in refrigeration (2 - 8 ° C) for several weeks. Figure 8 shows the results of the FIA of the residual ferrocyanide of both reactive inks. From Figure 8, the ratio of the signal to the reference (S / R) represents the relative amount of ferrocyanide in the reactive ink as compared to the ferrocyanide added as the reference in the FIA. In this way, the higher the S / R value of the FIA analysis, the greater the ferrocyanide in the reactive inks. It can be seen from Figure 8A that the value of S / R increases during the six week period for both control inks and for the reactive ink with added ferrocyanide. However, the reactive ink curve with added ferrocyanide has a slower increase in residual current over the six week period compared to the control curves. In Figure 8B, the S / R response curves of the control inks and the added ferrocyanide reactive ink are fused by comparison. For the first-order approximation (since the coefficients for the second-order terms of both second-order polynomials are very small), the rate of increase of residual current during six weeks in refrigeration is approximately 30% ([0.0918 - 0.0638] /0.0918 = 30%) lower for the reactive ink curve with added ferrocyanide than for the control curves. In this way, it can be understood from Figures 8A and 8B that the rate of the conversion of ferricyanide to ferrocyanide in the reactive ink is substantially decreased by the addition of the internal reference of ferrocyanide to the DEX reactive ink according to the invention. biosensor 102 of the invention. While the present invention has been described with reference to the details of the embodiments of the invention shown in the drawings, these details are not intended to limit the scope of the invention claimed in the appended claims.

Claims (20)

1. CLAIMS 1. A biosensor for the determination of the concentration of an analyte in a test sample, characterized in that it comprises: a mixture for the electrochemical reaction with an analyte; the mixture includes an enzyme, a mediator and an oxidizable species as an internal reference. A biosensor according to claim 1, characterized in that the internal reference is defined as the reduced form of a reversible redox coupling having a redox potential equal to or higher than that of the mediator. 3. A biosensor according to claim 1, characterized in that the mediator comprises 3-phenylimino-3H-phenothiazine. 4. A biosensor according to claim 3, characterized in that the internal reference comprises ferrocyanide. 5. A biosensor according to claim 4, characterized in that the ferrocyanide defining the internal reference and the mediator are oxidized at a first voltage potential and only the mediator is oxidized at a second voltage potential; the second voltage potential is smaller than the first voltage potential. 6. A biosensor according to claim 5, characterized in that the first voltage potential is approximately 400 V and the second voltage potential is approximately 100 mV. 7. A biosensor according to claim 1, characterized in that the mediator comprises ferricyanide. 8. A biosensor according to claim 7, characterized in that the internal reference comprises ferrocyanide. 9. A biosensor according to claim 1, characterized in that the mediator comprises ruthenium hexaamine. 10. A biosensor according to claim 9, characterized in that the internal reference comprises ferrocyanide. 11. A biosensor according to claim 10, characterized in that the enzyme comprises glucose oxidase. 12. A method for the use of a biosensor that includes a mixture of an enzyme, a mediator and an oxidizable species as an internal reference, the method is characterized in that it comprises the steps consisting of: applying a first voltage potential in a first period; provide a set delay period; apply a second voltage potential in a final period after the delay period; and wherein the first voltage potential and the second voltage potential are selectively provided to oxidize only the mediator or both the mediator and the internal reference. A method according to claim 12, characterized in that the step of applying a first voltage potential in a first period includes the step of applying a first high voltage potential, selected in the first period to oxidize the mediator and the reference internal A method according to claim 12, characterized in that the step of applying a first voltage potential in a first period includes the step of applying a first low voltage potential, selected in the first period to oxidize only the mediator. A method according to claim 12, characterized in that the step of applying a second voltage potential in a final period after the delay period includes the step of applying a second voltage potential selected to oxidize the mediator and the internal reference . 16. A method according to claim 12, characterized in that the step of applying a second voltage potential in a final period after the delay period includes the step of applying a second voltage potential selected to oxidize only the mediator. 17. A method according to claim 12, characterized in that the steps of applying the first voltage potential and applying the second voltage potential include the steps of applying a selected voltage potential in a range between 100 mV and 400 V. A method according to claim 12, characterized in that the steps of applying the first voltage potential and applying the second voltage potential include the steps of applying a first voltage potential selected in the first period to oxidize both the mediator and the internal reference; and apply a second voltage potential selected to oxidize only the mediator. 19. A method according to claim 12, characterized in that the biosensor includes a mediator comprising a 3-phenylimino-3H-phenothiazine and ruthenium hexaamine; and wherein the internal reference comprises ferrocyanide, and wherein the steps of applying the first voltage potential and applying the second voltage potential include the steps of applying a first and second voltage potential selected to oxidize only the mediator. 20. A method according to claim 12, characterized in that the steps of applying the first voltage potential and applying the second voltage potential include the steps of applying a first and second voltage potential selected to oxidize both the mediator and the internal reference; wherein the internal reference effectively secures a calibration intercept within a narrow range and the internal reference effectively maintains a calibration slope for the biosensor.