The research project proposes the development of laccase-based biosensors to monitor in situ the dynamic change in catecholamine concentration released from neuronal living cells under hypoxia conditions. The biosensor/cell construction incorporates living cells at the biosensor surface in direct contact with the enzymatic layer. Recently, electrochemical real-time monitoring of analytes within and/or released from living cells has provided important solutions to medical diagnosis and disease treatment. The importance of catecholamines (dopamine and norepinephrine) in the regulation of various physiological functions, turns these species into interesting analytical targets, with elevated levels leading to neurological or psychiatric disorders. The developed device is envisaged to be a suitable platform for cell growth and proliferation, with an embedded selective and sensitive mechanism based on the oxidation by the laccase enzyme of secreted dopamine and norepinephrine under external stimuli to the cell environment. The proposed mechanism has the potential to be applied for the external stimuli involvement investigation on exocytosis of neuronal living cells. This project comes in response to the need of in situ evaluation of the dynamic changes in neurochemical pathways, which remains to be fully explored.

Project Summary:

STAGE 1 - Development of laccase-based biosensors: Fabrication of Au/Ti/SiO₂/Si transducers and evaluation of laccase immobilization procedures on the electrode surface. Incorporation of carbon nanomaterials and metal nanoparticles aimed at signal amplification by direct electron transfer between the enzyme active center and the transducer surface. Incorporation of redox polymer films on the transducer and their evaluation as electrochemical mediators.

STAGE 2 - Optimization of laccase-based biosensors for catecholamine monitoring and live cell implantation: Optimization of the laccase-based biosensor for the quantification of catecholamines in cell culture medium and the study of interferents. Biocompatibility and cytotoxicity tests (MTS and LDH) of biosensor components. Construction of the biosensor/cell assembly by implanting the cells on the biosensor surface. This activity will be carried out in feedback with the results obtained in Stage 1.

STAGE 3 - Dynamic monitoring of the concentration of catecholamines released from neuronal cells, induced by hypoxia conditions: In situ quantification of catecholamines in live neuronal cells under conditions of hypoxia and chemical stimuli.

Scientific Results:

Development of Au/Ti/SiO2/Si transducers and evaluation of laccase immobilization procedures

• The first step comprised the development of Au/Ti/Si/SiO₂ transducers, for which, magnetron sputtering, a methodology successfully described as a procedure for the development of sensor surfaces with thin metal layers, was conducted on silicon wafers towards obtaining a thin adhesion layer of Ti (~10 nm) followed by a layer of Au (~200 nm). In order to investigate the electrochemical processes of dopamine, cyclic voltammetry was conducted. Even though the target analyte is electroactive, being oxidized at the electrode surface at 0.31V, with a reversible reduction process observed at 0.23V, distinct biosensing strategies are required to selectively monitor dopamine in complex media, thus hindering the signal from a wide range of electroactive biomolecules that might act as interferences.
• The laccase enzyme was hereby employed for the selective recognition of dopamine, given its catalytic activity for the oxidation of polyphenols at low overpotentials. A crucial parameter for the development of biosensing surfaces lies in the immobilization of the bioelement of recognition of choice, thus avoiding changes in their conformation, which might lead to differences in biological activity, rendering them unselective towards the targeted molecule. In order to ensure optimal biological activity of the laccase enzyme, two different methodologies were initially investigated in the construction of the proposed biosensors: (i) cross-linking with glutaraldehyde and (ii) covalent binding on self-assembled monolayers of short-chain thiol molecules. The Lac_GA/Au/Ti/SiO₂/Si and Lac/cys/Au/Ti/SiO₂/Si biosensors were investigated by cyclic voltammetry in buffer solution and amperometry upon successive additions of dopamine in different applied potential values ranging from -0.2V to 0.1V. For both immobilization methodologies, the signal recognition mechanism was attributed to the reduction of the product of the enzymatic reaction.

Incorporation of surface modifiers and dopamine detection

• The incorporation of carbon nanotubes and a conductive polymer, poly brilliant cresyl blue (pBCB) was investigated to increase the performance of laccase-based biosensors for dopamine recognition. CNT/Au/Ti/SiO₂/Si modified electrodes were constructed through drop-casting. In order to investigate the contribution of dye polymers on the final biosensor performance, electrochemical polymerization of BCB was conducted by cyclic voltamme Laccase was incorporated at the surface of both pBCB/CNT/Au/Ti/SiO₂/Si and CNT/Au/Ti/SiO₂/Si modified electrodes by cross-linking with GA.
• Fixed potential amperometry was employed to investigate the performance of the developed biosensors upon successive additions of dopamine. The applied potential corresponded to 0.1V, following the optimization previously conducted. The corresponding calibration curves were constructed based on amperometric data and illustrated the improved sensitivity values for laccase-based biosensors incorporating nanostructures. The Lac/CNT/Au/Ti/SiO₂/Si and Lac/pBCB/Au/Ti/SiO₂/Si biosensors presented sensitivity values of 0.430 µA µM^-1 cm^-2 and 0.302 µA µM^-1 cm^-2, respectively, with a 2-fold increase in performance when compared to laccase immobilized directly at the surface of the Au/Ti/SiO₂/Si transducer. The increased sensitivity obtained at the Lac/CNT/Au/Ti/SiO₂/Si biosensor (when compared to the Lac/pBCB/Au/Ti/SiO₂/Si biosensor) can be attributed to the increased charge transfer properties of the modified surface upon interaction with the oxidized dopamine after the enzymatic recognition takes place, most likely due to the compatibility of carbon-based surfaces with aromatic compounds. The final biosensor that contained both CNT and the pBCB presented the highest sensitivity values for dopamine detection, 0.5385 µA µM^-1 cm^-2, with a low deviation between consecutive measurements (RSD 2%) and a limit of detection of 0.233 µM. It is believed that the synergy between the increased surface area attributed to the use of carbon nanostructures, as well as the optimal conductivity properties of the dye polymer present as a modifying layer for laccase immobilization, support their use in the development of a sensitive and selective biosensor for monitoring dopamine in cell culture assays.

1. Daniela Oprea, Caroline G. Sanz, Madalina M. Barsan, Adrian T. Enache, "PC-12 Cell Line as a Neuronal Cell Model for Biosensing Applications", Biosensors 2022, 12(7), 500. doi: https://doi.org/10.3390/bios12070500

Conferences:

• Oral dissemination at the 1st Regional Meeting of the International Society of Electrochemistry (Prague, Czech Republic, 14-19 August, 2022).
• Oral dissemination at the 73rd Annual Meeting of the International Society of Electrochemistry (Online, 12-16 September, 2022).