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 . A sensor for detecting D-3-hydroxybutyrate in vivo, the sensor comprising:
a first portion configured to be positioned above a user's skin; and
a second portion configured to be transcutaneously positioned in the user's skin and in contact with the user's interstitial fluid to monitor the level of D-3-hydroxybutyrate in vivo, the second portion comprising:
i) a working electrode having an enzyme composition disposed thereon, the enzyme composition comprising:
a) nicotinamide adenine dinucleotide (phosphate) (NAD(P)+) or derivative thereof;
b) D-3-hydroxybutyrate dehydrogenase;
c) diaphorase;
d) an electron transfer agent;
e) a polymer,
wherein the NAD(P)+ or derivative thereof is not covalently bonded to the polymer; and
ii) a mass transport limiting membrane disposed over the enzyme composition,
wherein the working electrode is configured to generate a signal associated with the level of D-3-hydroxybutyrate,
wherein the mass transport limiting membrane limits flux of D-3-hydroxybutyrate to the working electrode, and
wherein the signal associated with the level of D-3-hydroxybutyrate increases linearly as a function of D-3-hydroxybutyrate concentration over a range of 0 mM to 10 mM.
2 . The analyte sensor of claim 1 , wherein the D-3-hydroxybutyrate dehydrogenase is covalently bonded to the polymer.
3 . The analyte sensor of claim 2 , wherein the diaphorase is covalently bonded to the polymer.
4 . The analyte sensor of claim 3 , wherein the electron transfer agent is covalently bonded to the polymer.
5 . The analyte sensor of claim 1 , wherein enzyme composition further comprises a crosslinker for immobilizing the enzyme composition onto a surface of the working electrode.
6 . The analyte sensor of claim 1 , wherein the mass transport limiting membrane comprises crosslinked polymers.
7 . The analyte sensor of claim 6 , wherein the polymers contain heterocyclic nitrogen groups.
8 . The analyte sensor of claim 6 , wherein the polymers comprise polyvinylpyridine.
9 . The analyte sensor of claim 6 , wherein the polymers comprise poly(4-vinylpyridine-co-styrene).
10 . The analyte sensor of claim 7 , wherein the mass transport limiting membrane further comprises a hydrophilic or hydrophobic modifier.
11 . The analyte sensor of claim 1 , wherein the mass transport limiting membrane is a polyurethane or polyether urethane membrane.
12 . The analyte sensor of claim 1 , wherein the signal and D-3-hydroxybutyrate concentration exhibit an R 2 value of at least 0.99 over a range of 0 mM to 10 mM.
13 . The analyte sensor of claim 1 , wherein the signal decays no more than 8% when exposed to a given D-3-hydroxybutyrate concentration for 45 hours.