The development and application of an engineered direct electron transfer enzyme for continuous levodopa monitoring

Abstract

Levodopa, the primary treatment for Parkinson’s Disease, has a narrow therapeutic window further complicated by the lack of real-time feedback, primarily due to the absence of an enzyme specific to levodopa. We addressed this by developing a novel direct electron transfer type (DET) enzyme, copper dehydrogenase (CoDH), engineered from an extremophile derived multicopper oxidase (MCO), for use in a continuous levodopa sensor. By introducing mutations into the type 2 and type 3 copper ligand histidine residues, the enzyme drastically decreased its oxidase activity while enhancing DET activity with the electrode. Using this developed CoDH, a chronoamperometric levodopa sensor was constructed, which was minimally affected by environmental changes, or by interferents, including levodopa metabolites, adjunct medications, and common plasma and interstitial fluid components. A miniaturized levodopa sensor was constructed and was able to detect levodopa as low as 138 nM, suggesting its future application for in vivo subcutaneous measurement.