During inflammation, cells produce and release nitric oxide (NO), a metabolite which can be used as a biomarker to monitor the progression of inflammatory diseases1. Common methods imply the use of Griess method or paramagnetic electron resonance (EPR) for indirect NO measurement in a solution. However, all these methods cannot provide real time monitoring, limiting the investigation of drug response and the clinical translation. The use of electrochemical sensors can overcome this issue; however, the selectivity of this sensing tool could be critical if not specifically designed for the biological use2.
As sensor's working electrodes, platinum wires were used bare or modified with polymers and/or Carbon Black nanomaterial3. Two different modifications were performed: an electropolymerization with poly-o-phenylenediammine (p-OPD/PPD), applying +700 mV vs an Ag/AgCl reference electrode and a physical adsorption on the wire surface with CB dispersion in NafionTM by drop casting and oven drying (200°C for 2 minutes). The sensors were calibrated with known concentration (0 to 100 µM) of a NO donor molecule, the SNAP (s-nitroso-n-acetylpenicillamine), by applying a potential of +865 mV. As common biological interferences, Ascorbic acid (AA), L-glutammine (Glu) and hydrogen peroxide H2O2 were tested. Nitric oxide was measured in real time in 2D chondrocyte culture for 48h in presence and absence of 10 ng/ml IL1β, by applying the same potential vs Ag/AgCl RE directly in cell culture plate.
p-OPD and CB were homogenously distributed on sensor surface. The coating with p-OPD showed lower background noise for AA and none for Glu but considerable for H2O2. Contrarily, the coating with CB-Nafion strongly decreased all the interferences. The real time monitoring of NO in chondrocytes showed significant difference in signal between the groups. The use of CB coating increased the analytical performance in terms of selectivity and reproducibility during real time monitoring in inflamed biological system.
Discussion and conclusions: Real time monitoring of NO release by electrochemical sensor during the inflammation in vitro and eventually in vivo could help to determine the progression/status of widespread pathologies or infections from pathogens in biological fluids and cell culture medium. However, we provide evidence that in a complex system such as cell culture medium, it is of paramount importance to properly modify the active surface of the sensor to avoid the interference with analytes not of interested, such as the large production of hydrogen peroxide. Herein, we prove how the modification with CB-Nafion does not limit the sensor performance while increasing the selectivity for the NO.
Acknowledgements: AO Foundation, Innosuisse funding scheme.
1 Sharma, J., Al-Omran, A. & Parvathy, S. Role of nitric oxide in inflammatory diseases. Inflammopharmacology 15, 252-259 (2007).
2 Rocchitta, G. et al. Enzyme biosensors for biomedical applications: Strategies for safeguarding analytical performances in biological fluids. Sensors 16, 780 (2016).
3 Arduini, F. et al. Carbon black as an outstanding and affordable nanomaterial for electrochemical (bio) sensor design. Biosensors and Bioelectronics 156, 112033 (2020).