Electrochemical analyte sensors using thermostable soybean peroxidase
First Claim
1. An electrochemical sensor, comprising:
- (a) an electrode;
(b) a hydrogen peroxide sensing layer on the electrode, comprising a thermostable peroxidase and non-leachable redox centers for transferring electrons between the electrode and the peroxidase; and
(c) a second enzyme capable of catalyzing the production of hydrogen peroxide in the presence of an analyte, the second enzyme being disposed on the electrode proximate the sensing layer but electrically insulated from the sensing layer;
wherein the sensor operates continuously at 37°
C. for 100 hours or more with less than 10% drop in the sensor'"'"'s output.
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Abstract
A sensor for the detection and measurement of an analyte in a biofluid. The sensor includes two enzymes. One type of sensor measures the concentration of hydrogen peroxide using a thermostable peroxidase enzyme that is immobilized in a redox hydrogel to form a sensing layer on a working electrode. This sensor also includes a hydrogen peroxide-generating second enzyme which is insulated from the redox hydrogel and electrode. This second enzyme generates hydrogen peroxide in response to the presence of an analyte or analyte-generated compound. The second enzyme may be insulated from the electrode by placement of an electrically insulating layer between the sensing layer and the second enzyme layer.
Alternatively, the second enzyme is immobilized in an inorganic polymeric matrix, preferably made using a sol-gel polymerization process. Such matrices include those made of silica. Often, the second enzyme is stabilized by immobilization in a sol-gel. Further stabilization of polyelectrolytic enzymes can be obtained by immobilizing the enzyme with a polyelectrolytic polymer in the sol-gel matrix.
198 Citations
38 Claims
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1. An electrochemical sensor, comprising:
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(a) an electrode;
(b) a hydrogen peroxide sensing layer on the electrode, comprising a thermostable peroxidase and non-leachable redox centers for transferring electrons between the electrode and the peroxidase; and
(c) a second enzyme capable of catalyzing the production of hydrogen peroxide in the presence of an analyte, the second enzyme being disposed on the electrode proximate the sensing layer but electrically insulated from the sensing layer;
wherein the sensor operates continuously at 37°
C. for 100 hours or more with less than 10% drop in the sensor'"'"'s output. - View Dependent Claims (2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24)
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25. A composition comprising:
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an enzyme; and
a polymer binding the enzyme; and
a matrix comprising silicon and oxygen atoms, and having the bound enzyme-polymer immobilized therein. - View Dependent Claims (26, 27, 28, 29, 30, 31)
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32. An electrochemical analyte sensor comprising:
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an electrode;
a hydrogen peroxide sensing layer comprising a thermostable soybean peroxidase immobilized on the electrode with a redox polymer;
a hydrogen peroxide generating layer comprising an enzyme which catalyzes a reaction of the analyte whereby hydrogen peroxide is produced, and further comprising a silicone; and
an analyte-transport controlling layer, selectively permeable to oxygen. - View Dependent Claims (33, 34)
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35. An electrochemical analyte sensor comprising:
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(a) an electrode;
(b) a first layer placed on the electrode and comprising a thermostable peroxidase immobilized on the electrode with a redox polymer;
(c) a second layer placed on the first layer and comprising a film of cellulose acetate;
(d) a third layer placed on the second layer and comprising an oxidase which catalyzes the production of hydrogen peroxide in the presence of an analyte; and
(e) a fourth layer placed on the third layer and comprising a film of cellulose acetate.
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36. An electrochemical hydrogen peroxide sensor comprising:
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(a) a non-corroding electrode; and
(b) a thermostable peroxidase immobilized on the non-corroding electrode;
the sensor having no leachable components, and capable of operation at temperatures of 37°
C. or higher with less than 2% loss of sensitivity per hour of continuous operation.
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37. A method for the electrochemical analysis of hydrogen peroxide, the method comprising the steps of:
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(a) generating an electrical signal by an electrode in the presence of hydrogen peroxide, the electrode comprising a thermostable peroxidase coimmobilized with a non-leachable redox compound on a non-corroding electrode; and
(b) correlating the generated electrical signal with the presence of hydrogen peroxide in the sample.
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38. A method for continuous measurement of an analyte, comprising the steps of:
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(a) placing a sensor on or in a body fluid or tissue, the sensor comprising a thermostable peroxidase coimmobilized with a non-leachable redox compound onto a non-corroding electrode and a second enzyme immobilized on the electrode, the second enzyme catalyzing a reaction which generates hydrogen peroxide in the presence of a specified analyte; and
(b) correlating an electrical signal generated by the electrode with an amount of analyte present in the body fluid or tissue.
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Specification