NAD(P)- dependent responsive enzymes, electrodes and sensors, and methods for making and using the same
NADP-dependent oxidoreductase compositions, and electrodes, sensors and systems that include the same. Analyte sensors include an electrode having a sensing layer disposed thereon, the sensing layer comprising a polymer and an enzyme composition distributed therein. The enzyme composition includes nicotinamide adenine dinucleotide phosphate (NAD(P) + ) or derivative thereof; an NAD(P) + -dependent dehydrogenase; an NAD(P)H oxidoreductase; and an electron transfer agent comprising a transition metal complex.
1 . An electrochemical analyte sensor configured for partial insertion below a subject's skin surface, the sensor comprising:
a portion configured to be positioned above the subject's skin surface and an inserted portion configured to be positioned below the subject's skin surface, wherein the inserted portion comprises:
a non-conductive material layer;
a first conductive layer on at least a portion of the non-conductive material, wherein the first conductive layer is a working electrode;
an enzyme composition deposited on a surface of the first conductive layer, wherein the enzyme composition comprises:
a) nicotinamide adenine dinucleotide (phosphate) (NAD(P)+) or derivative thereof;
b) D-3-hydroxybutyrate dehydrogenase;
c) diaphorase;
d) a polymeric redox mediator; and
a mass transport limiting membrane disposed over the enzyme system to limit flux of β-hydroxybutyrate to the working electrode,
wherein the NAD(P)+ is not covalently bonded to the polymeric redox mediator.
2 . The electrochemical analyte sensor of claim 1 , further comprising a first insulation layer disposed on at least a portion of the first conductive layer.
3 . The electrochemical analyte sensor of claim 2 , further comprising a second conductive layer positioned on top of at least a portion of the first insulation layer.
4 . The electrochemical analyte sensor of claim 3 , further comprising a second insulation layer positioned on at least a portion of the second conductive layer.
5 . The electrochemical sensor of claim 4 , further comprising a third conductive layer positioned on top of at least a portion of the second insulation layer.
6 . The electrochemical sensor of claim 1 , wherein the first conductive layer comprises a conductive polymer, carbon, platinum-carbon, or gold.
7 . The electrochemical sensor of claim 5 , wherein the second conductive layer is a reference electrode.
8 . The electrochemical sensor of claim 7 , wherein the third conductive layer is a counter electrode.
9 . The electrochemical sensor of claim 1 , further comprising a second conductive layer positioned on the non-conductive material layer, wherein the second conductive layer is coplanar to and spaced apart from the first conductive layer.
10 . The electrochemical sensor of claim 9 , further comprising a third conductive layer disposed on a side opposite from the non-conductive material layer.
11 . The electrochemical sensor of claim 8 , wherein the portion configured to be positioned above the user's skin comprises contact portions of the working electrode, the reference electrode, and the counter electrode.
12 . The electrochemical sensor of claim 11 , wherein traces connect electrodes to the contact portions.
13 . The electrochemical sensor of claim 11 , wherein the contact portions are configured for connection with sensor electronics.
14 . The electrochemical sensor of claim 12 , further comprising a via providing an avenue through the non-conductive material layer, wherein at least one trace connects an electrode and respective contact on opposite sides of the non-conductive material layer.
15 . The electrochemical sensor of claim 1 , wherein the enzyme composition deposited on the surface of the first conductive layer as a plurality of spots, the plurality of spots having a discontinuous or continuous perimeter.
16 . The electrochemical sensor of claim 15 , wherein the plurality of spots have a discontinuous perimeter.
17 . The electrochemical sensor of claim 15 , wherein the plurality of spots have a continuous perimeter.
18 . The electrochemical sensor of claim 15 , wherein the plurality of spots form a square, rectangle, or circle when viewed from above.
19 . The electrochemical sensor of claim 15 , wherein all or a portion of the surface of the first conductive layer is covered by the enzyme composition.
20 . The electrochemical sensor of claim 19 , wherein all of the surface of first conductive layer is covered by the enzyme composition.
21 . The electrochemical sensor of claim 1 , wherein the enzyme composition deposited on the surface of the first conductive layer is in the form of an array of a plurality of spots spaced apart from each other.
22 . The electrochemical sensor of claim 1 , wherein the mass transport limiting membrane comprises polyvinylpyridine.
23 . The electrochemical sensor of claim 1 , wherein the mass transport limiting membrane comprises poly(4-vinylpyridine-co-styrene).