Amperometric biosensors based on redox enzymes
First Claim
1. An amperometric biosensor for the measurement of the concentration of creatine, creatinine and/or sarcosine comprising:
- an electrode comprising a surface;
an electronically active mediator deposited on the surface of the electrode; and
a population of enzymes comprising either sarcosine dehydrogenase or sarcosine dehydrogenase and at least one hydrolase enzyme deposited on the surface of the electrode, wherein the population of enzymes is covalently linked.
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Abstract
The invention includes various prototypical amperometric biosensors for the quantification of biological substrates such as fructose, creatinine, creatine, and sarcosine, and the methods for producing these biosensors. Also included in the invention is a minaturized version of the biosensing devices. The components of these prototypical biosensors are immobilized on a self-assembled monolayer (SAM) comprising chemisorbed alkanethiols. The deposition of an amphiphilic lipid layer to these systems increases the stability and activity of the resultant biosensor and enhances the rejection of many interferents. An additional feature of the invention is the co-deposition of the components of the sensor via a novel detergent dialysis protocol. The invention features two particular biosensor systems. One embodiment involves fructose dehydrogenase as the redox/sensor enzyme and fructose as the substrate/analyte. Another embodiment involves the measurement of the substrates/analytes, creatinine, creatine and sarcosine, using sacrosine dehydrogenase as the redox/sensor enzyme, with the involvement of creatinine amidohydrolase and/or creatine amidinohydrolase in the reaction pathway.
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Citations
27 Claims
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1. An amperometric biosensor for the measurement of the concentration of creatine, creatinine and/or sarcosine comprising:
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an electrode comprising a surface;
an electronically active mediator deposited on the surface of the electrode; and
a population of enzymes comprising either sarcosine dehydrogenase or sarcosine dehydrogenase and at least one hydrolase enzyme deposited on the surface of the electrode, wherein the population of enzymes is covalently linked. - View Dependent Claims (2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16)
a metal wire with a working end plated with a noble metal or a carbon fiber;
an electrode surface at the working end upon which sarcosine dehydrogenase and at least one hydrolase enzyme are immobilized to form a working electrode;
an encasement containing the working electrode drawn to a tip of about 1-20 micrometers in diameter;
a Ag/AgCl wire inserted into the encasement wherein the Ag/AgCl wire serves both as a reference and counter electrode;
and an electrolyte filler inserted into the encasement.
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6. The amperometric biosensor of claim 5, wherein the hydrolase enzyme is either creatine amidinohydrolase or creatinine amidohydrolase or a mixture of both creatine amidinohydrolase or creatinine amidohydrolase.
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7. The amperometric biosensor of claim 1, wherein the electrode is either metallic or non-metallic.
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8. The amperometric biosensor of claim 7, wherein the metallic electrode is gold, silver, platinum or palladium.
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9. The amperometric biosensor of claim 7, wherein the non-metallic electrode comprises carbon.
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10. The amperometric biosensor of claim 1, further comprising a chemisorbed lipophilic layer deposited on the surface of the electrode.
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11. The amperometric biosensor of claim 10, wherein the lipophilic layer comprises a thiol.
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12. The amperometric biosensor of claim 1, further comprising an amphiphilic lipid deposited on the surface of the electrode.
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13. The amperometric biosensor of claim 12, wherein the amphiphilic lipid is a phospholipid.
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14. The method of using an amperometric biosensor to measure the concentration of creatine, creatinine and/or sarcosine comprising:
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assembling the amperometric biosensor of claim 1;
providing a sample; and
measuring the current produced by oxidation of any sarcosine, creatine and/or creatinine present in the sample.
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15. The method of claim 14, further comprising adding a stabilizer protein to the amperometric biosensor prior to measuring the current.
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16. The method of claim 14, wherein the electrode is metallic or non-metallic.
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17. A method of preparing an amperometric biosensor capable of measuring the concentration of creatine, creatinine and/or sarcosine comprising:
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adding a covalent linking agent to a mixture of sarcosine dehydrogenase and at least one hydrolase enzyme to form a population of substantially covalently linked enzymes;
depositing the population of covalently linked enzymes on an electrode surface; and
adding an electronically active mediator to the population of enzymes either before or after the enzymes are deposited on the electrode surface.- View Dependent Claims (18, 19)
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20. An amperometric biosensor for the measurement of the concentration of creatine, creatinine and/or sarcosine comprising:
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an metallic electrode comprising a surface;
a chemisorbed lipophilic layer deposited onto the electrode surface;
an electronically active mediator added to the lipophilic layer; and
a population of redox enzymes comprising sarcosine dehydrogenase and a hydrolase enzyme deposited onto the lipophilic layer. - View Dependent Claims (21, 22, 23, 24, 25, 26)
assembling the amperometric biosensor of claim 20;
providing a sample; and
measuring the current produced by oxidation of any sarcosine, creatine and/or creatinine present in the sample.
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26. The biosensor of claim 20, wherein the hydrolase enzyme is either creatine amidinohydrolase or a mixture of creatine amidohydrolase and creatinine amidohydrolase.
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27. A method of preparing an amperometric biosensor capable of measuring the concentration of creatine, creatinine and/or sarcosine comprising:
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preparing a metallic electrode surface by adding a lipophilic layer via chemisorption;
depositing a mixture of sarcosine dehydrogenase and at least one hydrolase enzyme to form a population of immobilized enzymes on the electrode surface; and
adding an electronically active mediator to the population of enzymes either before or after the enzymes are deposited on the electrode surface.
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Specification