An Amperometric Biosensor Based on a Bilayer of Electrodeposited Graphene Oxide and Co-Crosslinked Tyrosinase for L-Dopa Detection in Untreated Human Plasma

Abstract

L-Dopa, a bioactive compound naturally occurring in some Leguminosae plants, is the most effective symptomatic drug treatment for Parkinson’s disease. During disease progression, fluctuations in L-DOPA plasma levels occur, causing motor complications. Sensing devices capable of rapidly monitoring drug levels would allow adjusting L-Dopa dosing, improving therapeutic outcomes. A novel amperometric biosensor for L-Dopa detection is described, based on tyrosinase co-crosslinked onto a graphene oxide layer produced through electrodeposition. Careful optimization of the enzyme immobilization procedure permitted to improve the long-term stability while substantially shortening and simplifying the biosensor fabrication. The effectiveness of the immobilization protocol combined with the enhanced performances of electrodeposited graphene oxide allowed to achieve high sensitivity, wide linear range, and a detection limit of 0.84 μM, suitable for L-Dopa detection within its therapeutic window. Interference from endogenous compounds, tested at concentrations levels typically found in drug-treated patients, was not significant. Ascorbic acid exhibited a tyrosinase inhibitory behavior and was therefore rejected from the enzymatic layer by casting an outer Nafion membrane. The proposed device was applied for L-Dopa detection in human plasma, showing good recoveries.

A Critical Overview of Enzyme-Based Electrochemical Biosensors for L-Dopa Detection in Biological Samples

“…With respect to mere crosslinking, the presence of an additional inert protein allows the mitigation of the deactivation of the enzyme and improves the mechanical stability of the immobilized membrane. Regarding tyrosinase immobilization, crosslinking (without the inert protein) [65,66] and co-crosslinking (with BSA as an inert protein) [67][68][69][70] have both been employed in the presence of glutaraldehyde (GLU) as a reticulation agent. The methods developed have often been based on consecutive steps to deposit the proteins and GLU on the electrode [65,67,69].…”

“…The employment of a deposition solution made of both tyrosinase and GLU allows the shortening of the airdrying time to 12 h [66]. The immobilization time can be further reduced to 2 h [68] and even just to 20 min [70] when tyrosinase, GLU, and BSA are simultaneously present in the co-crosslinking solution. In the latter case, an additional step has been devised to improve the mechanical stability of the immobilized layer, consisting of dipping the electrode in a GLU solution for 15 min and then air drying for 30 min.…”

“…The operational stability of the above sensors has been tested on short time scales extended mainly to one or two weeks [65,67] or has not been reported at all [66]. The best performances have been achieved by Cembalo et al [70], whose device after three weeks showed a sensitivity of around 80% of the initial value, and Pinho et al [69], who reported a stability of more than a month. In the latter case, however, a very laborious multistep procedure was employed for tyrosinase immobilization, with timescales of more than 24 h. First, the gold surface was modified with amine functionalities following the chemisorption of cysteamine; the electrode was then left immersed in a GLU solution for 2 h at room temperature.…”

“…Recently, GO was employed by Cembalo et al [70] for the realization of an LD biosensor with a linear range of 1-210 µM, a sensitivity of 3.21 µA/mM, and a limit of detection of 0.84 µM. The novelty of the proposed sensor is the technique adopted for GO deposition.…”

A Paradigm Shift in the Management of Patients with Parkinson’s Disease

“…Moreover, efficient management of PD depends on the detection of nonmotor symptoms and biomarkers. Wearable devices capable of real-time measurement of levodopa levels, combined with systems that capture motor symptoms, could significantly enhance the available information for optimizing treatment decisions [72,73]. The incorporation of continuous blood pressure measurements into these systems could provide valuable insights into autonomic dysfunction, aiding in the effective management of orthostatic hypotension [74,75].…”