Electrolysis: processes – compositions used therein – and methods – Electrolytic analysis or testing – Involving enzyme or micro-organism
Reexamination Certificate
1999-01-28
2003-05-20
Tung, T. (Department: 1743)
Electrolysis: processes, compositions used therein, and methods
Electrolytic analysis or testing
Involving enzyme or micro-organism
C204S403010, C204S403140
Reexamination Certificate
active
06565738
ABSTRACT:
BACKGROUND OF THE INVENTION
1. Field of the Invention
This invention relates to a biosensor for the determination of the concentration of an analyte in a biological fluid, such as, for example, a biosensor for the determination of alanine aminotransferase activity in whole blood.
2. Discussion of the Art
Alanine aminotransferase (hereinafter “ALT”) is an enzyme that is found primarily in the liver and is released into the blood when the liver is damaged. ALT is an important analyte, the concentration of which is elevated in almost all disorders of the liver. ALT measurement is used to help diagnose liver disorders or liver damage, such as from hepatitis, toxins, or adverse reactions to ethical drugs. The number of estimated deaths in the United States in 1994 attributed to adverse drug reactions was over 100,000, making adverse drug reaction the fifth leading cause of death in the United States. The estimated cost of adverse drug reactions in the United States is over 77 billion dollars per year. A single ALT measurement does not indicate whether the liver damage is progressing. Consequently, serial ALT measurements are frequently used to aid in the diagnosis of a liver disorder and to follow its progress.
ALT assays of the prior art have typically employed blood collected from an individual through a needle in the arm connected to an evacuated tube and processed by centrifugation to acquire serum or plasma. The serum or plasma is then evaluated for ALT activity by a trained operator, usually by means of a test system employing an instrument that is normally at least as large as a personal computer. The reagents used in these ALT assays generally exhibit limited stability at ambient and elevated temperatures. Therefore, the reagents must typically be stored at a location removed from the instrument and under refrigerated conditions. Assays that employ liquid reagents typically require precise addition of the reagents to the serum or plasma and mixing the added reagents with the serum or plasma to obtain accurate ALT measurements.
Because of the complexity of ALT assays currently available, trained operators must conduct the assays. Moreover, the large size of the analyzers prohibit system portability. For these reasons, the patient must travel to a patient-care facility to have an ALT assay run. Some patients must have their ALT levels monitored regularly by their physician for months, or even years.
The field of self-monitoring is well established in the area of diabetes. Self-monitoring devices for determining blood glucose levels have been available to patients since the mid 1980's. These devices use a fingerstick to obtain whole blood and are very simple to operate. It would be desirable to develop an ALT assay that is simple enough for patients to run by themselves without the need to travel to a patient-care facility
U.S. Pat. No. 5,705,045 describes a biosensor capable of measuring both ALT and aspartate aminotransferase (AST) simultaneously. The biosensor contains two sets of electrodes, each set being sensitive to a different liver enzyme. Each electrode consists of a screen-printed layer containing carbon, enzyme, and binder. Overlying this layer is a screen-printed layer containing reagents and binder. In an assay employing this biosensor, a biological fluid containing ALT or AST is placed on the biosensor. The ALT or AST reacts with the reagents to produce peroxide or NADH, which is then oxidized, thereby resulting in a current that is proportional to the concentration of whichever liver enzyme is being measured. The patent describes two different chemical reactions for measuring liver enzymes. One chemical reaction produces peroxide and the other produces NADH. Any system that uses NADH to detect ALT requires NAD, which is expensive and unstable. A system that uses peroxide to detect ALT does not need NAD and would therefore be less expensive and more stable. The sensor has a cover that forms a capillary zone to move a sample from a sample application zone to the electrodes. The sensor has reagents mixed into the binder. The reagents and the binder dissolve upon exposure to the sample. In such systems, the analyte measurement must be carried out immediately after exposure of the electrode to the sample because the product of the chemical reaction and carbon diffuse away from the surface of the electrode, resulting in greatly reduced signals at long reaction times. ALT concentrations in humans are very low. The ALT reaction pathway in U.S. Pat. No. 5,705,045 requires a reaction time of several minutes to produce a measurable amount of H
2
O
2
. Therefore, this method of ALT measurement will not operate with a soluble binder system.
There is no mention of whole blood as a suitable sample in U.S. Pat. No. 5,705,045. If whole blood were to be used as a sample, the sample would clot over the sensor during the reaction period. A whole blood sample would have to be treated with an anticoagulant at a location remote from the biosensor. Capillary blood from a fingerstick could not be used because there is no means to easily add an anticoagulant to it. This deficiency would prevent the sensor from being used for home testing because capillary blood is the only sample accessible to the patient.
The measurement of ALT and AST by a biosensor was also described by Cooper et al., in Analytica Chimica Acta, 245 (1991) 57-62. Cooper employs an electrode made from platinized carbon. The sample of serum is diluted with a liquid reagent prior to the determination. Liquid reagents are undesirable because they require mixing with the sample prior to initiation of the assay. Liquid reagents are also typically less stable than dry reagents. Adding liquid reagent to a sample also dilutes the sample, which results in a lower ALT concentration in the sample, which is more difficult to measure than is a higher concentration of ALT in an undiluted sample. The addition of the liquid reagent to the sample must be carried out in a quantitative manner to obtain an accurate result. The system is complex and, therefore, would not be suitable for home use.
Currently, there is no ALT assay available for patients that they can carry out themselves. For existing ALT assays, there are many time-consuming and manual steps required before a result can be reported. Usually the patient must travel to a physician's office, hospital, or patient-care facility to have the assay run. Many ALT assays require serum as the sample, so a person trained in drawing blood is needed to obtain the sample from the patient by means of an unpleasant venipuncture procedure. Then, the blood sample must be processed by centrifugation to obtain the serum or plasma sample. Many ALT assays use liquid reagents, which must be stored under refrigerated conditions. When the assay is to be run, the liquid reagents must be added to the test system. Because of the complexity of the assay or test system, a trained operator is usually required. Furthermore, the concentration of ALT in blood is low, with the result that an accurate determination of ALT concentration is difficult.
SUMMARY OF THE INVENTION
This invention provides a biosensor for determining the concentration of an analyte, such as, for example, ALT, in a sample of biological fluid, such as, for example, whole blood, comprising:
(a) a base layer;
(b) a detecting layer comprising a reference electrode and a working electrode, the surface of the electrically conductive portion of the working electrode being sufficiently smooth so that determination of the concentration of an analyte present in a low concentration, e.g., less than 1 mM, can be made;
(c) a layer overlying said electrodes, said layer comprising dried reagents; and
(d) an anticoagulant disposed in such a location that it will prevent the sample from coagulating during the determination.
The base layer provides mechanical support for all the other layers in the biosensor. The detecting layer enables a parameter of the product released by the chemical reaction of the analyte, e.g., ALT, to be transformed in
Cousineau Kristen L.
Henning Timothy P.
Abbott Laboratories
Noguerola Alex
Weinstein David L.
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