Continuous Levodopa Sensor Employing Engineered Novel Direct Electron Transfer Type Enzyme
Authors: Probst David , Batchu Kartheek , Satomura Takenori , Sode Koji 
Published: December 2024

Introduction

Parkinson's disease (PD) is a neurodegenerative condition marked by the loss of dopaminergic neurons in the substantia nigra, leading to reduced dopamine production and symptoms like bradykinesia and tremors. The gold standard treatment for PD is levodopa, a dopamine precursor, which is orally administered and can cross the blood-brain barrier, where it is converted into dopamine. A critical challenge of levodopa therapy is maintaining a stable plasma and cerebral concentration of levodopa, which could be solved by the development of a continuous levodopa monitor. Analogous to the success of continuous glucose monitoring in diabetes mellitus management, continuous monitoring of levodopa levels in PD may potentially help clinicians to more accurately develop levodopa therapies, adjust oral therapies, or even integrate with continuous levodopa infusion systems in the future; these systems have already been introduced in Europe. Despite these recent advancements and the potential benefit of improved monitoring, no FDA-approved levodopa sensor exists today, primarily due to the lack of a stable and selective molecular recognition element.

In this presentation, we introduce our proposed solution to the challenge of creating an enzymatic sensor for continuous, real-time levodopa monitoring, by employing an engineered direct electron transfer (DET) type enzyme with extremely high stability and specificity toward levodopa.

Methods

We prepared an electrochemical levodopa sensor employing a novel engineered enzyme that specifically recognizes levodopa. The enzyme is immobilized on the gold electrode using a self-assembled monolayer (SAM). The sensor was operated using a combination of electrochemical techniques, including chronoamperometry, open circuit potential (OCP), and transient OCP. We investigated the sensor’s performance using both buffer solutions and human venous serum spiked with levodopa across the cerebrospinal fluid and blood therapeutic ranges.

Result and discussion

To acknowledge the capability of the engineered enzyme, which can transfer electrons directly to the electrode, we developed a DET-type levodopa sensor that does not require oxygen or synthetic electron acceptors. The levodopa sensor exhibited excellent sensitivity and specificity across the entire levodopa therapeutic window in both buffer solutions and spiked human serum samples. Thereafter, a levodopa microsensor was also constructed and characterized using gold microwire with a diameter of 76 µm. To leverage the specific benefits of OCP sensing, such as the sensor’s signal independence from the electrode size, we roughened the gold wire to increase enzyme density, thereby enhancing the sensor’s sensitivity and dynamic range for monitoring. We investigated the impact of potential interferents of the levodopa sensor, including 3-0-methyldopa, carbidopa, and associated levodopa intermediates/metabolites, revealing that there was minimal interferent bias in the sensor’s signals.

Conclusion

We developed a highly reliable enzymatic levodopa microsensor, employing an engineered DET-enzyme, which showed high specificity toward levodopa with sufficient sensitivity and dynamic range that covers the physiological range, both in buffer solution and in ex-vivo spiked human serum. Given its small size, sensitivity, and tolerance towards artifacts, we have determined that the sensor is suitable for subcutaneous implantation, thus enabling it for future in-vivo, real-time levodopa monitoring in interstitial fluid.