NAD(P)-dependent responsive enzymes, electrodes and sensors, and methods for making and using the same

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.