Electrochemical biosensors based on NAD(P)-dependent dehydrogenase enzymes
Inventors
Pei, Jianhong • Young, Chung Chang
Assignees
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Publication Number
US-8500990-B2
Publication Date
2013-08-06
Expiration Date
Abstract
A biosensor for measuring an analyte in a liquid sample includes a working electrode having a dispensed reagent thereon wherein the dispensed reagent contains an enzyme capable of catalyzing a reaction involving the analyte, a mediator that is considered an enzyme inhibitor, and an enzyme co-factor where the working electrode provides a stable and sensitive response even when the biosensor is stored at ambient conditions for a period of time selected from the group consisting of at least 3 months, 12 months and two years or more.
Core Innovation
A method for determining information indicative of the level of an analyte in a liquid sample uses a working electrode having a reagent matrix disposed thereon. The reagent matrix is formed from a dispensing reagent containing an enzyme capable of catalyzing a reaction involving the analyte, an enzyme inhibitor mediator selected from meldola's blue, 4-methyl-1,2-benzoquinone, and 2,6-dichloroindophenol, and an enzyme co-factor. The enzyme co-factor is present in the dispensing reagent at about 0.01% to about 3.0% (w/w) and the mediator is present at about 0.05% to about 3.0% (w/w).
The approach generates an electrical output from the reagent matrix and measures the electrical output to determine the level of the analyte in the liquid sample using information comprising the measured electrical output. A biosensor includes the working electrode and a reference electrode, where the working electrode comprises the reagent matrix formed from the dispensing reagent containing the enzyme, the mediator considered an enzyme inhibitor, and the enzyme co-factor. The working electrode produces a stable and sensitive response after storage at ambient conditions for at least 3 months, 12 months, and two years or more.
In particular implementations based on NAD(P)-dependent dehydrogenase enzymes, the biosensor measures the concentration of an analyte in a liquid sample without calibrating the working electrode. A method of making the biosensor forms a working electrode and a reference electrode on a substrate, formulates the dispensing reagent with the mediator and enzyme co-factor at defined concentration ranges and ratios enabling stable response at ambient storage, dispenses the reagent onto the working electrode, and evaporates the dispensing reagent to form the reagent matrix.
Claims Coverage
The independent claims define electrochemical methods and biosensors centered on a working electrode with a reagent matrix using a mediator considered an enzyme inhibitor, an enzyme co-factor, and an enzyme capable of catalyzing an analyte reaction. Across the independent claims, the inventive core includes 3 inventive feature areas: mediator selection and reagent-matrix composition ranges, ambient-stability performance requirements for the working electrode, and electrical output measurement using a reference electrode, with an additional NAD(P)-dependent implementation that measures analyte concentration without calibrating the working electrode and a method of making the biosensor.
Stable, sensitive ambient storage working electrode reagent matrix
A working electrode having a reagent matrix formed from a dispensing reagent containing an enzyme capable of catalyzing a reaction involving the analyte, a mediator considered an enzyme inhibitor, and an enzyme co-factor, wherein the mediator is selected from meldola's blue, 4-methyl-1,2-benzoquinone, and 2,6-dichloroindophenol, the enzyme co-factor having a concentration of about 0.01% to about 3.0% (w/w) and the mediator having a concentration of about 0.05% to about 3.0% (w/w), and the working electrode provides a stable and sensitive response when stored at ambient conditions for at least 3 months or longer; generating an electrical output from the reagent matrix; measuring the electrical output; and determining the level of the analyte in the liquid sample using information comprising the measured electrical output.
Biosensor with reference electrode and ambient-stable response
A working electrode having a reagent matrix formed from a dispensing reagent containing an enzyme capable of catalyzing a reaction involving the analyte, a mediator considered an enzyme inhibitor, and an enzyme co-factor, wherein the mediator is selected from meldola's blue, 4-methyl-1,2-benzoquinone, and 2,6-dichloroindophenol, the enzyme co-factor having a concentration of about 0.01% to about 3.0% (w/w) and the mediator having a concentration of about 0.05% to about 3.0% (w/w), and the working electrode provides a stable and sensitive response when stored at ambient conditions for a period of time selected from at least 3 months, 12 months, and two years or more; and a reference electrode.
NAD(P)-dependent dehydrogenase biosensor measuring analyte without calibrating
A working electrode having a reagent matrix formed from a dispensing reagent containing mediator considered an enzyme inhibitor, NAD(P)-dependent dehydrogenase enzymes, and an enzyme co-factor, wherein the mediator is selected from meldola's blue, 4-methyl-1,2-benzoquinone, and 2,6-dichloroindophenol, the enzyme co-factor having a concentration of about 0.01% to about 3.0% (w/w) and the mediator having a concentration of about 0.05% to about 3.0% (w/w), and the working electrode is capable of measuring the concentration of an analyte in a liquid sample without calibrating the working electrode; and a reference electrode.
Method of making an ambient-stable biosensor by forming reagent matrix
Forming a working electrode and a reference electrode on a substrate; formulating a dispensing reagent containing an enzyme capable of catalyzing a reaction involving the analyte, a mediator considered an enzyme inhibitor, and an enzyme co-factor, wherein the mediator is selected from meldola's blue, 4-methyl-1,2-benzoquinone, and 2,6-dichloroindophenol, the enzyme co-factor having a concentration of about 0.01% to about 3.0% (w/w) and the mediator having a concentration of about 0.05% to about 3.0% (w/w), and the ratio of the enzyme co-factor and the mediator enables the working electrode to have a stable response when stored at ambient conditions for 3 months or longer; dispensing a quantity of the dispensing reagent onto the working electrode; and evaporating the dispensing reagent to form a reagent matrix on the working electrode.
Across the independent claims, the claim coverage is directed to electrochemical methods and biosensors in which a working electrode with a reagent matrix formed from a dispensing reagent includes a mediator considered an enzyme inhibitor selected from the named mediator group, an enzyme co-factor at specified weight-percent ranges, and an enzyme capable of catalyzing an analyte reaction, including NAD(P)-dependent dehydrogenase enzymes. The independent claims further require stable and sensitive response under ambient storage conditions for defined periods, measured electrical output with a reference electrode for biosensor claims, and an NAD(P)-dependent implementation that measures analyte concentration without calibrating the working electrode. A related independent claim covers making such a biosensor by forming electrodes, formulating the dispensing reagent with defined mediator/co-factor composition, dispensing reagent onto the working electrode, and evaporating to form the reagent matrix.
Stated Advantages
Provides a stable and sensitive response when the working electrode is stored at ambient conditions for at least 3 months or longer.
Provides a stable and sensitive response even when stored at ambient conditions for selected time periods including at least 3 months, 12 months and two years or more.
Capable of measuring the concentration of an analyte in a liquid sample without calibrating the working electrode.
Documented Applications
Determining information indicative of the level of an analyte in a liquid sample using an electrochemical working electrode reagent matrix.
Measuring an analyte in a liquid sample using a biosensor that includes a working electrode with a reagent matrix and a reference electrode.
NAD(P)-dependent dehydrogenase biosensor measurement of analyte concentration in a liquid sample without calibrating the working electrode.
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