News Column

"Oxidizable Species as an Internal Reference in Control Solutions for Biosensors" in Patent Application Approval Process

June 17, 2014

By a News Reporter-Staff News Editor at Life Science Weekly -- A patent application by the inventors Beer, Greg P. (Cassopolis, MI); Wu, Huan-Ping (Granger, IN), filed on January 29, 2014, was made available online on June 5, 2014, according to news reporting originating from Washington, D.C., by NewsRx correspondents (see also Bayer HealthCare LLC).

This patent application is assigned to Bayer HealthCare LLC.

The following quote was obtained by the news editors from the background information supplied by the inventors: "More specifically, this invention relates to the biosensors that are used to measure the amount of analytes in bodily fluids. Optical methods are often used for making such measurements, but the present invention relates to improvements in electrochemical biosensors. While the method to be described herein can be applied to measurement of other analytes, including cholesterol, urea, creatinine, and creatine, measuring glucose in whole blood is of particular interest. Although the description here will emphasize the inventions application to measuring glucose, it should be understood that the invention has broader applications.

"The invention relates to an electrochemical instrument in which a potential is applied to electrodes in contact with a biological sample and reagents. The resulting current is measured while the analyte is reacting with the reagents, and then correlated with the amount of an analyte in the sample. Such instruments are referred to as amperometric, in contrast with coulometric instruments that measure the total charge in coulombs produced from reaction of the sample by integrating the current over the entire measurement time. The amperometric instruments have an advantage in that they are less volume and time dependent. They do not wait for the entire volume of the analyte to be reacted, but only take measurements of the analyte by sampling the reaction rate at a predetermined time.

"Many designs for such biosensors have been described in the art, for example, published U.S. Patent Application 2001/0042683. The electrodes are generally described as the working electrode and as the counter electrode. The electrodes are in contact with a solid layer containing reagents that oxidize the analyte in the sample, such as glucose oxidase, and mediators that reoxidize the reduced enzyme. The reduced mediator itself is oxidized at the working electrode, which produces a measurable current. This current is used to calculate the amount of glucose in the sample being tested, since it is an indirect measure of the oxidation of glucose in the sample. The reactions may be described by the following steps:

" (Gluconic Acid, Gluconolactone)


" e.sup.-

"Where E.sub.oxid and are oxidized and reduced forms of the redox center of the enzyme and Med.sub.oxid and are the oxidized and reduced forms of the mediator.

"For measuring glucose, the enzyme may be glucose oxidase and the mediator ferricyanide. Measuring other analytes will employ suitable enzymes and mediators. Typical combinations of enzyme, mediator and analyte are listed in Table 1.

"TABLE-US-00001 TABLE 1 Selected Substrates, Enzyme and Mediator Systems Analyte Enzyme Mediator Glucose Glucose Oxidase Ferricyanide Glucose Glucose Dehydrogenase Ferricyanide Cholesterol Cholesterol Oxidase Ferricyanide Lactate Lactate Oxidase Ferricyanide Uric Acid Uricase Ferricyanide Alcohol Alcohol Oxidase Phenylenediamine

"In order to assure accurate measurements, control solutions containing known amounts of glucose are used to verify that the instrument is operating properly. The composition of control solutions has been the subject of a number of patents and publications. Representative are U.S. Pat. Nos. 3,920,580; 4,572,899; 4,729,959; 5,028,542 and 5,605,837; WO 93/21928; WO 95/1.3535; and WO 95/13536. While control solutions containing blood serum have been used, more recent patents have been concerned with replacing serum-based control solutions with solutions free of serum, since serum-free solutions are more consistent and stable than those containing serum. The control solution should contain a known concentration of glucose in a serum-like matrix to determine the accuracy of both the enzymatic biosensor and the potentiostat meter. It will be evident that the composition must be stable over lengthy periods of storage before use.

"Control solutions should serve the purpose of checking the glucose monitoring system's functioning, but at the same time they should be identified and separated from the readings of real blood samples. This is because the control solutions contain known amounts of glucose and provide readings that have no therapeutic purpose. If the control solutions cannot be identified and their responses separated from those of the blood samples by the test meter, glucose readings of the control solutions will be included in the history of the glucose measurements, which could lead to wrong interpretation of a patient's diabetic condition. Or, if a control solution is substituted for a blood sample, it may be mistakenly considered by a physician as indicating a need to change treatment. Furthermore, since the temperature response of the control solutions is different from that of the blood samples, temperature compensation for measurements made at temperatures other than 25'DC will be less accurate if a test meter cannot distinguish between blood samples and control solutions. Therefore, it is highly desirable that the glucose monitoring system automatically detect and identify the control solutions in order to separate the glucose readings of control solutions from those of the blood samples, and to provide separate temperature compensation to both the blood samples and the control solutions.

"There have been several patents describing methods of identifying the control solutions through various mechanisms. In U.S. Pat. No. 5,723,284, electrochemical measurement of glucose in blood is discussed. The '284 patent proposed to modify the control solutions, changing the ratio of current readings taken from two oxidation periods separated by a rest period. The meter would recognize that a control solution was being measured and take appropriate action to prevent the results from being included in the blood sample results. The '284 patent also teaches that the control solution should be buffered in a pH range of 4.8 to 7.5 to be effective.

"Another method for determining whether a control solution or a blood sample is being measured for its glucose content is disclosed in U.S. Published Application 2002/0139692A1. An index is determined, that relates the decline of electrical current to the nature of the sample being tested.

"U.S. Pat. Nos. 5,620,579 and 5,653,863, proposed to begin the test of a sample by providing, an initial positive potential pulse for a short period in order to reoxidize any prematurely reduced mediator. Such an initial pulse was referred to as a 'burnoff period'.

"When a potential is applied across the working, and counter electrodes and a liquid sample is introduced to the sensor, the dry reagents are rehydrated by the liquid sample and current begins to flow, typically increasing to a peak and then declining over the 'burn period,' usually about ten seconds in length. During this period the previously reduced mediator is reoxidized to reduce the bias towards incorrect high results. If a full amount of sample is not present, additional error may be introduced since all of the reagents may not become available for reaction or the working and counter electrodes might not be in complete contact with sample, thus reducing the current during the 'burn' period.

"After the burn period has been completed, a rest period is provided at a lower potential or at no potential (open circuit). During this rest period the glucose oxidation reaction continues to take place and the mediator is reduced. Then, a constant potential is applied again between the working and counter electrodes and the current is measured for a short period, typically about two to ten seconds. The current is initially high, but it declines rapidly as diffusion of the mediator begins to control. At a predetermined time, the measured current is used to determine the glucose content of the sample.

"Adding an internal reference compound is a common practice in analytical chemistry to provide a quantitative reference signal. This working principle has been used in a recent published patent application No. WO 2005/078118, where an internal reference is added to the reagent system to achieve some formulation purpose.

"In WO2004/040286A1, it is proposed that the control solution include a reducing substance chosen from uric acid, bilirubin, ascorbic acid, methylene blue, Bis(2-hydroxyethyl)iminotris(hydroxymethyl)methane, N,N-bis(2-hydroxyethyl)-2-aminoethane sulfonic acid, and acetaminophen, thus changing the ratio of current readings taken from two oxidation periods separated by a rest period and enabling the control solution to be identified.

"The present inventors have sought an improved method of distinguishing control solutions from biological samples. Their methods are described in detail below."

In addition to the background information obtained for this patent application, NewsRx journalists also obtained the inventors' summary information for this patent application: "This invention provides a method for distinguishing a control solution from a biological sample during the operation of an electrochemical meter through a quantitative index. In one embodiment, the invention is a control solution which includes known amounts of glucose, a buffer system with a suitable pH value, and an internal reference compound. The internal reference compound is added to the control solution to identify the control solution by the glucose monitoring system. The invention provides a method of detecting the presence of the internal reference compound, calculating the quantitative index, and identifying that a control solution is being tested.

"In one embodiment, the analyte in the biological sample is glucose in whole blood and the mediator is potassium ferricyanide. The internal reference compound is oxidizable at a potential higher than the potential required to oxidize the mediator, which is used to measure the oxidation of analyte. The internal reference has a predetermined concentration in the control solution along with a predetermined concentration of glucose. The glucose-related mediator and the internal reference are selectively oxidized at the electrode by different potentials (low and high). When glucose is the analyte, the internal reference compound should be oxidizable at a potential of at least 100 my higher than the potential used to re-oxidize the reduced mediator.

"Internal reference compounds useful for measuring glucose in control solutions include any species oxidizable at the electrode at an appropriate potential such as organo-metallic compounds, coordination compounds, and organic amine compounds. The amount of the internal reference compound used is related to the amount of glucose in the control solution. Preferably, the amount of the internal reference compound is chosen so that the control solution can be recognized when the glucose in the control solution is the maximum required to test the electrochemical glucose meter. Alternatively, the amount of the internal reference compound can be varied in proportion to the amount of glucose.

"Comparing measurements made at low and high potentials provides a method to detect the internal reference compound and thus the control solution. When the measurement protocol employs two periods separated by a rest period, the high and low voltages can be applied in either period or both. A potential is applied, to a sensor that has received the control solution. The current produced at a potential capable of oxidizing the internal reference compound is compared with the current produced when a potential capable of oxidizing only the analyte glucose), but not the internal reference compound. The ratio between the two measured currents, designated the Differential Index (DI), provides a means for distinguishing the control solution from a liquid sample lacking the internal reference compound.

"DI=i.sub.high volt/i.sub.low volt

"where i.sub.high volt and i.sub.low volt are the currents measured at high and low voltages. At the higher voltage, both the internal reference compound and the reduced mediator are oxidized, while at the lower voltage only the mediator is oxidized,

"A DI value of about 1 indicates that the sample lacks the internal reference compound and is presumed to be a biological sample, while a DI value significantly greater than 1, preferably at least about 1.5, indicates that the sample is a control solution.

"In another embodiment, the invention is an internal reference compound suitable for indicating the presence of a control solution being used to test the accuracy of an electrochemical biosensor/potentiostat system. Where the analyte is glucose in whole blood the internal reference compound maybe an oxidizable organo-metallic compound, a coordination compound, or an organic amine.

"One result of the present invention is the improvement of the consistency of the current measured and the accuracy of the resulting analysis. If high and low potentials are applied during the same oxidation period, such as the burn and read periods used in oxidation of glucose, it is less likely that external factors, such as sample movement or environmental temperature will affect the Differential Index. Multiple readings of the current developed at high and/or low voltages can improve the accuracy of the results. Also, when the presence of a control solution has been determined, a temperature correction algorithm particular to the control solution can be applied. By using different temperature algorithms for the control solution and the biological sample, (e.g., whole blood), clinical results can be improved and tighter control range values can be assigned.


"FIG. 1 is an exploded view of a biosensor according to one embodiment.

"FIG. 2 is an assembled view of the biosensor of FIG. 1.

"FIG. 3 is one plot of potential versus time for a burn period.

"FIG. 4 is one plot of current versus time for the potential applied as FIG. 3.

"FIG. 5 is a cyclic voltammogram described in Example 1.

"FIG. 6 is a cyclic voltammogram described in Example 2.

"FIG. 7 is a cyclic voltammogram described in Example 3.

"FIG. 8 is a cyclic voltammogram described in Example 4.

"FIG. 9A-E, illustrate potential sequences described in Example 5.

"FIG. 10a-b are cyclic voltammograms described in Example G.

"FIG. 11 is a plot of the current versus time obtained in Example 7.

"FIG. 12 is a plot of the current versus time obtained in Example 8."

URL and more information on this patent application, see: Beer, Greg P.; Wu, Huan-Ping. Oxidizable Species as an Internal Reference in Control Solutions for Biosensors. Filed January 29, 2014 and posted June 5, 2014. Patent URL:

Keywords for this news article include: Ions, Chemistry, Biosensing, Electrolytes, Legal Issues, Ferricyanides, Bioengineering, Electrochemical, Ferric Compounds, Bionanotechnology, Nanobiotechnology, Inorganic Chemicals, Bayer HealthCare LLC, Enzymes and Coenzymes.

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Source: Life Science Weekly

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