Development of an Engineered Direct Electron Transfer Enzyme for Continuous Levodopa Monitoring

Background

Parkinson’s Disease (PD) is a chronic neurodegenerative disease that affects millions of people worldwide. It is characterized by the loss of dopaminergic neurons in the substantia nigra and the presence of α-synuclein aggregations forming Lewy bodies in the neurons. Levodopa (L-DOPA) is the principal treatment for PD and effectively alleviates motor symptoms. However, its narrow therapeutic window can result in severe side effects such as nausea, dyskinesia, or a rebound of symptoms due to improper dosing. This issue highlights the need for real-time monitoring of levodopa levels.

Unlike continuous glucose monitoring (CGM) devices widely used in diabetes management, continuous levodopa monitoring in PD management has not yet been realized. According to the authors of this study, the development of a continuous levodopa monitoring device could not only aid in optimizing the dosage for PD patients but also significantly enhance our understanding of levodopa pharmacokinetics, thereby improving disease management. This research introduces an innovative continuous levodopa sensor to address the current limitations.

Source of the Article

This study was published in the journal npj Biosensing, under the title “The development and application of an engineered direct electron transfer enzyme for continuous levodopa monitoring.” The research was conducted by Kartheek Batchu, David Probst, Takenori Satomura, John Younce, and Koji Sode, affiliated with the Joint Department of Biomedical Engineering at the University of North Carolina at Chapel Hill and North Carolina State University, the University of Vermont College of Medicine, the Division of Engineering at Fukui University, and the Department of Neurology at UNC Chapel Hill. The paper was officially published in 2025.

Research Design and Experimental Workflow

1. Overall Design of the Study

To achieve continuous levodopa monitoring, the research team developed a novel enzyme, copper dehydrogenase (CODH), based on the direct electron transfer (DET) mechanism. This enzyme was derived from a multicopper oxidase (MCO) found in a hyperthermophilic archaeon. Using site-directed mutagenesis, the authors modified the Type 2 and Type 3 copper ligand residues, significantly reducing oxidase activity while improving direct electron transfer capacity with the electrode surface.

2. Methods and Experimental Details

a) Engineering of the Enzyme

The research team began with the MCO model and engineered the enzyme through specific mutations at residues His396 and His459 (e.g., H396A/H459A). This resulted in significant changes to the enzyme’s redox mechanism. The engineered MCO was renamed as “Copper Dehydrogenase” (CODH). Directed evolution removed the Type 2 and Type 3 copper sites, while retaining the Type 1 copper (T1 copper) as the site for direct electron transfer with the electrode instead of oxygen as the terminal electron acceptor.

The authors confirmed the preservation of the T1 copper site using UV-Vis absorption spectroscopy and excluded any activity from the T2/T3 copper centers. The mutated CODH was immobilized onto a gold electrode surface using a dithiobis-succinimidyl hexanoate (DSH)-based self-assembled monolayer (SAM), optimizing sensor precision.

b) Validation of Enzyme Activity and Stability

The oxidase activity of the engineered enzyme, measured through the oxidation of ABTS substrate, was reduced to below 1.5% of the original MCO. Further direct electron transfer experiments demonstrated that the engineered CODH maintained stable catalytic currents under deoxygenated conditions. The peak signals corresponding to a 360 µM levodopa concentration dropped only by 21%, even when exposed to atmospheric oxygen.

c) Development of the Electrochemical Sensor

The initial prototype sensor was constructed using a three-electrode system: a gold disc electrode (GDE) as the working electrode, a silver/silver chloride reference electrode, and a platinum counter electrode. The research team then miniaturized the sensor using a 76.2 μm gold microwire as the working electrode and adopted a two-electrode system, optimizing it for subcutaneous insertion.

d) Performance Testing and Evaluation

Electrochemical measurements were conducted in levodopa solutions with a concentration range of 0–55 µM. The miniaturized levodopa sensor exhibited excellent performance in 100 mM phosphate buffer (pH 7.0) with a limit of detection (LOD) of 138 nM and a sensitivity of 0.0042 µA/µM. Additionally, interference testing with substances such as dopamine or hydroxytyrosol showed that background signals deviated by less than 10%, except for 3-o-Methyldopa, which showed a slight deviation above 10%.

3. Impact of Environmental Factors and Long-Term Storage Stability

The study demonstrated that the sensor’s electrochemical signal remained stable over a pH range of 6–8 and temperatures from room temperature (25 °C) to body temperature (37 °C). Additionally, the sensor retained more than 95% of its initial sensitivity during a 21-day storage period at 4 °C.

4. Clinical Implications and Future Experimental Plans

The research team plans to integrate the sensor with subcutaneous applications in clinical PD patients. Future experiments will focus on developing biocompatibility-enhancing membrane layers and noise-reduction algorithms to improve sensor stability and functionality in vivo. A key objective is to combine this technology with existing continuous levodopa infusion systems, paving the way for a closed-loop treatment system for PD.

Significance and Highlights of the Research

This work represents the first reported development of a DET-type multicopper oxidase enzyme that does not rely on oxygen as a terminal electron acceptor. It is also the first study to construct a levodopa sensor based on such an enzyme. Key highlights include:

1. High Specificity for Levodopa Detection: The engineered CODH exhibited excellent specificity for levodopa, significantly reducing interference compared to sensors using tyrosinase or direct oxidation methods.

2. Innovative Direct Electron Transfer Mechanism: The removal of the oxygen reduction half-reaction allowed the T1 copper site to directly interact with the gold electrode, making the sensor independent of dissolved oxygen fluctuations.

3. Miniaturized Design and Rigorous Interference Testing: The sensor was successfully miniaturized and demonstrated excellent practicality in interference testing with 18 compounds under conditions that closely mimic clinical environments.

4. Long-Term Stability for Continuous Monitoring: The sensor exhibited stable performance over three weeks in laboratory conditions, highlighting its potential for real-time monitoring and dosage optimization.

Conclusion

This study presents a revolutionary electrochemical method for real-time levodopa monitoring through the development of a DET-type enzyme—copper dehydrogenase (CODH). It demonstrated high stability and specificity. This innovative technology not only provides a foundation for personalized treatment of PD patients but also establishes a framework for designing continuous monitoring systems for other drugs. With further research and clinical evaluation, the described technology holds the potential to become a breakthrough in PD management, improving the quality of life for patients.