Briefly, a sensor for a continuous biological monitor is provided for measuring the level of a target analyte for a patient. The sensor has a working wire and a reference wire, where the working wire has an analyte limiting layer that passes more than 1 in 1000 analyte molecules from the patient to the an enzyme layer. The enzyme layer has an enzyme entrapped in a polyurethane cross-linked with acrylic polyol. As free electrons are generated, a conductor transfers the electrons to the biological monitor. In some cases, the sensor may be constructed without the use of any expensive platinum.

The invention provides amperometric analyte sensor systems comprising one or more electrodes designed to monitor in vivo levels of 3-hydroxybutyrate (and optionally glucose as well) in order to facilitate the management of diabetic ketoacidosis. The invention further includes compositions, elements and methods useful with such amperometric analyte sensor systems.

Described is a method for the production of 3-buten-2-one comprising the enzymatic conversion of 4-hydroxy-2-butanone into 3-buten-2-one by making use of an enzyme catalyzing 4-hydroxy-2-butanone dehydration, wherein said enzyme catalyzing 4-hydroxy-2-butanone dehydration is (a) a 3-hydroxypropiony-CoA dehydratase (EC 4.2.1.116), (b) a 3-hydroxybutyryl-CoA dehydratase (EC 4.2.1.55), (c) an enoyl-CoA hydratase (EC 4.2.1.17), (d) a 3-hydroxyoctanoyl-[acyl-carrier-protein] dehydratase (EC 4.2.1.59), (e) a crotonyl-[acyl-carrier-protein] hydratase (EC 4.2.1.58), (f) a 3-hydroxydecanoyl-[acyl-carrier-protein] dehydratase (EC 4.2.1.60), (g) a 3-hydroxypalmitoyl-[acyl-carrier-protein] dehydratase (EC 4.2.1.61), (h) a long-chain-enoyl-CoA hydratase (EC 4.2.1.74), or (i) a 3-methylglutaconyl-CoA hydratase (EC 4.2.1.18). The produced 3-buten-2-one can be further converted into 3-buten-2-ol and finally into 1,3-butadiene.

A working electrode for a subcutaneous sensor for use with a continuous biological monitor for a patient is disclosed. The working electrode includes a conductive substrate and a carbon-enzyme layer on the conductive substrate. The carbon-enzyme layer includes a polyurethane or silicone crosslinked with an acrylic polyol, and an enzyme fully entrapped by the polyurethane or silicone crosslinked with the acrylic polyol. The enzyme is selected according to a biological function to be monitored. The carbon-enzyme layer also includes a carbon material. The carbon-enzyme layer is electrically conductive and facilitates a generation of either peroxide or electrons within the carbon-enzyme layer responsive to reacting the enzyme with a target biologic from blood of the patient.

The present disclosure provides analyte sensors including one or more NAD(P)-dependent enzymes and an internal supply of NAD(P) for the detection of an analyte. The present disclosure further provides methods of using such analyte sensors for detecting one or more analytes present in a biological sample of a subject, and methods of manufacturing said analyte sensors.

Engineered D-β-hydroxybutyrate dehydrogenases (BHBDhs) are disclosed. In some embodiments, the BHBDhs are derived from enzymes which are putative homologues of A. faecalis-derived BHBDh, including from thermophilic bacteria and radiation-resistance bacteria. These BHBDhs may also comprise additional mutations to increase their catalytic activity. Further, BHBDhs modified to include an NAD-binding loop, so as to tightly bind an NAD.sup.+ co-factor, are also disclosed.

Briefly, a carbon working electrode is described that has a plastic substrate of polyethylene, polypropylene, polystyrene, polyvinyl chloride, or polylactic acid, and may be formed into an elongated wire. The carbon material coats the plastic substrate, and may be, for example, graphene, diamagnetic graphite, pyrolytic graphite, pyrolytic carbon, carbon black, carbon paste, or carbon ink, which is aqueously dispersed in an elastomeric material such as polyurethane, silicone, acrylates or acrylics. Optionally, selected additives may be added to the carbon compound prior to it being layered onto the plastic substrate. These additives may, for example, improve electrical conductivity or sensitivity, or act as a catalyst for target analyte molecules.

Briefly, a sensor for a continuous biological monitor is provided that has a working electrode with an enhanced carbon-enzyme layer that in one embodiment is made by mixing an aqueous polyurethane emulsion with an acrylic polyol emulsion to make a base emulsion. An enzyme and carbon materials are added to the base emulsion, which is applied to the working electrode and cured. The carbon materials may include carbon and graphite to provide strength, as well as graphene or pyrolytic graphite to provide a desirable electrical resistance for the carbon-enzyme layer. Optionally, other additives can be added to the base emulsion prior to application, such as hydophiles, cross linkers, adding imodeoesters, hydroxysuccimide, carboldilite, melamines, epoxies, benzoyl peroxide or dicumyl peroxide.

A working electrode for a subcutaneous sensor for use with a continuous biological monitor for a patient is disclosed. The working electrode includes a conductive substrate and an enzyme layer on the conductive substrate. The enzyme layer includes an enzyme, and the enzyme selected according to a biological function to be monitored. A hydrophobic material cross-linked with an acrylic polyol is included. The enzyme is fully entrapped in the cross-linked hydrophobic material with the acrylic polyol.

Briefly, a sensor for a continuous biological monitor is provided that has a working electrode with an enhanced enzyme layer that in one embodiment is made by mixing an aqueous polyurethane emulsion with an acrylic polyol emulsion to make a base emulsion. An enzyme is added to the base emulsion, which is applied to the working electrode and cured. Optionally, other additives can be added to the base emulsion prior to application, such as hydrophiles, cross linkers, adding imodeoesters, hydroxysuccimide, carboldilite, melamines, epoxies, benzoyl peroxide or dicumyl peroxide.