Icon Legend

This session is not in your schedule.

This session is in your schedule. Click again to remove it.

Device Technologies and Biomedical Robotics

Undergraduate Advances in Device Technologies

Development of Disposable Electrochemical Levodopa Sensor for Management of Parkinson's Disease

Saturday, October 26, 2024

9:00 AM - 9:15 AM EST

Location: Room 327

Presenting Author(s)

Ameen R. Zafiruddin (he/him/his)

Student
The University of North Carolina at Chapel Hill
Raleigh, North Carolina, United States

Co-Author(s)

LS

Luke A. Shehee

Student
The University of North Carolina at Chapel Hill, United States
DP

David Probst

Post Doctoral Research Associate
The University of North Carolina at Chapel Hill, United States
KB

Kartheek Batchu

Graduate Student
The University of North Carolina at Chapel Hill, United States

Primary Investigator(s)

Koji Sode, PhD (he/him/his)

Principle Investigator
Joint Department of Biomedical Engineering, The University of North Carolina at Chapel Hill and North Carolina State University, Chapel Hill, NC 27599, USA
Chapel Hill, North Carolina, United States

Introduction: Parkinson’s Disease (PD) is a progressive neurodegenerative disorder that leads to the depletion of dopaminergic neurons in the substantia nigra. This depletion interferes with the dopamine-dependent communication projection to the dorsal striatum, giving rise to movement abnormalities including bradykinesia and rest tremors. These motor symptoms, termed parkinsonism, severely impact the overall quality of life for those with PD, reported to be the second most common neurodegenerative disorder at more than 10 million cases worldwide. While there isn’t a definitive cure for PD, a widely-used treatment is the administration of levodopa, a precursor to dopamine that is used to pass the blood-brain barrier in order to supplement decreasing endogenous dopamine levels.
Healthcare professionals commonly adjust levodopa therapy based on patients’ self-reported symptoms, though a challenge of levodopa therapy is maintaining the correct dosage; too much causes dyskinesia while too little allows parkinsonism to persist. Unfortunately, patients are often unable to distinguish between dyskinesia and parkinsonism and the bounds for this therapeutic window change as PD progresses. As such, a method for real-time monitoring of levodopa in PD patients is necessary. While several analytical methods are used for detecting levodopa, these assays are often resource- and time-intensive and are impractical for clinical application. An alternative approach is the use of disposable electrochemical-based sensors, which have the potential to allow for rapid and low-cost levodopa measurement. In this project, we explore various approaches to the concept with the goal of developing a biosensor strip for on-site, near real-time monitoring of plasma levodopa levels.

Materials and

Methods: Initial experiments utilized screen-printed carbon electrodes (SPCE) that were cleaned via CV in NaOH. 1 mg/mL of enzyme was deposited on the SPCE, dried in an incubator, and immobilized with 2.5% glutaraldehyde. Experiment 1 utilized a mutant multi-copper oxidase. Levodopa was added to a cell with the electrode in 8 increments from 0.3 to 55 uM, each stirred for 2 minutes and CA at 0.3 V. Experiment 2 utilized laccase instead, except with 4 voltages ranging from -0.3 to 0.3 V and 5 levodopa increments from 0 to 25 uM.
Following experiments utilized gold-sputtered electrodes (GSE) that were cleaned roughened via CV. Either 0.5 or 2.0 mg/mL of a tyrosinase dilution was deposited on the GSE and dried in a refrigerator. Experiment 3 involved 8 combinations of high-low conditions undergoing CA at -0.3 V: 2.0 or 0.5 mg/mL enzyme, 60- or 5-second equilibrium time (ET), and 0.5 or 10.0 uM levodopa. Experiment 4 utilized the same set-up, but removed the 5-second ET, changed to -0.4 V, and had 3 levodopa increments from 0 to 5 uM. Experiment 5 involved 2 cycles of CV of Experiment 4’s combinations from -0.5 to 0.5 V at 25 mV/s.
Final experiments utilized glassy carbon electrodes (GCE), polished and cleaned via CV in KOH, under CA at -0.4 V after a 60-second ET with a 5 levodopa increments from 0 to 25 uM stirred for 30 seconds. Experiment 6 had 2.0 mg/mL of tyrosinase drop-casted and immobilized with glutaraldehyde. Experiment 7 had no enzyme deposited.

Results, Conclusions, and Discussions: The developed sensor functions under the principle of chronoamperometry (CA). Immobilized copper-containing enzyme on the electrode surface oxidizes levodopa into dopaquinone. Once a negative potential is applied, the electrode begins to reduce dopaquinone back into levodopa via electron-transfer. This transfer results in a measurable negative current for a gradient of concentration, a cross-section of which is used to develop a current-to-concentration calibration curve.
Initial attempts with the SPCEs were unsuccessful given the poor response and correlation. These variations are attributed to poor implementation of the novel enzyme and inappropriate CA voltage. As such, laccase was utilized on voltages ranging from -0.3 V to 0.3 V. The laccase SPCE CA trial utilizing -0.3 V showed a desirable negative trend on strong correlation coefficient. Following additional study and analysis of other attempts in literature, gold electrodes utilizing tyrosinase were prominent. As such, the following attempts utilized GSEs. An initial trial with various conditions showed poor results and was attributed to error in the experimental design or electrode configuration. Following a second more refined trial using GSE with similarly poor results, a CV was performed and it was discovered that the significant reduction of oxygen on gold at voltages above -0.4 V overwhelmed any levodopa signal. As such, the last series of trials utilized GCE with tyrosinase at -0.4 V and showed promising concentration correlation with a good response and sensitivity. The 60 seconds wait time had the best response and lowest error, which can be attributed to the fact that the CA reaches a steady-state current given time for the reduction and oxidation of dopaquinone and levodopa to equilibrate. The blank trial of the GCE electrode configuration was performed to eliminate any experimental error.
Overall, significant difficulties were met during implementation of the sensor though the final results show a well founded response that can be improved on. Immediate future steps include additional testing with GCEs to implement a mesoporous carbon and switch back to the mutant multi-copper oxidase. Long-term efforts would translate this back to SPCEs and perform interference testing with human serum.

Acknowledgements (Optional): I would like to thank the Joint Department of Biomedical Engineering at the University of North Carolina at Chapel Hill and North Carolina State University, along with Dr. Koji Sode, Dr. David Probst, Kartheek Batchu, and the rest of the project’s internal and external collaborators for their invaluable guidance and knowledge throughout this project. I would also like to give a special thanks for Luke Shehee for staying by my side for every experiment and helping me build my passion for this project and the field.