Innovative Molecular Imprinting Sensor for Quick, Non-Invasive Cortisol Monitoring in Fish Welfare

Abstract

The assessment of fish welfare is crucial to prevent economic losses in aquaculture and ensure reliable results in research. A quick, non-invasive device to measure cortisol levels in fish farm water facilitates welfare evaluation and corrective actions when compromised. To address this need, an innovative sensor was developed using screen-printed carbon electrodes (SPCEs) functionalized with reduced graphene oxide/Prussian blue nanocubes (rGO/PBNCs) for direct selective detection of cortisol. A molecularly imprinted polymer (MIP) was synthesized on rGO/PBNCs/SPCEs by electropolymerization (ELP) of pyrrole in the presence of cortisol. The polymerization solution was prepared by adding cortisol (5 mM) and pyrrole (0.3 M) to a DMF/PBS (1:4) solution (pH 7.4). Following ELP, the electrodes were washed with PBS, and pyrrole overoxidation was used to extract cortisol from the polymer matrix. For comparison purposes, a non-imprinted polymer (NIP) was also fabricated. The electrodes were characterized using scanning electron microscopy (SEM) and Raman spectroscopy to assess their morphological and chemical features. Under optimized conditions, the sensor showed a linear range from 0.1 nM to 0.1 mM in artificial saltwater. This sensor combines simplicity and affordability while providing reliable detection of chemical and biological compounds.

Keywords: European seabass; aquaculture; screen-printed carbon electrode (SPCE); welfare assessment; zebrafish.

Figure 1

Schematic representation of the different steps necessary for the cortisol MIP sensor fabrication, calibration, and sample measurement.

Figure 2

CV measurements were performed at various stages of the fabrication process for the MIP and NIP. (A,B) The CV response following the immobilization of the rGO/PBNC nanocomposites and subsequent formation of the MIP and its non-imprinted control. (C) The CV response of the MIP after the removal of the target molecule, and (D) the corresponding response for the NIP.

Figure 3

Raman spectroscopy for (A) SPCE and SPCE/rGO/PBNCs, (B) SPCE/rGO/PBNCs/MIP, SPCE/rGO/PBNCs/NIP, (C) SPCE/rGO/PBNCs/MIP and SPCE/rGO/PBNC/MIP after treatment with overoxidation, and (D) SPCE/rGO/PBNC/NIP and SPCE/rGO/PBNC/NIP after treatment with overoxidation.

Figure 4

SEM images for (A) SPCE, (B) SPCE/rGO/PBNCs, (C) SPCE/rGO/PBNCs/MIP and (D) SPCE/rGO/PBNCs/NIP materials.

Figure 5

Electrochemical characterization of the MIP-based biosensor using SWV (A), along with the corresponding calibration curve (B) and the corresponding calibration curve for the NIP sensor (C), obtained by incrementally increasing the cortisol concentration in buffer.

Figure 6

Electrochemical characterization of the MIP-based biosensor using SWV (A) along with the corresponding calibration curve (B), obtained by incrementally increasing the cortisol concentration in PBS (red dots) and artificial saltwater (green dots).

Figure 7

Electrochemical characterization of the MIP-based biosensor using SWV (A), with measurements performed for different cortisol concentrations in buffer without the use of a redox probe. (B) The corresponding calibration curve obtained for MIP.

Figure 8

Relative current intensity data extracted from the SWV plots for cortisol standard solutions and other possible interfering species.