Electrochemical Quantification of H2O2 Released by Airway Cells Growing in Different Culture Media

Abstract

Quantification of oxidative stress is a challenging task that can help in monitoring chronic inflammatory respiratory airway diseases. Different studies can be found in the literature regarding the development of electrochemical sensors for H2O2 in cell culture medium to quantify oxidative stress. However, there are very limited data regarding the impact of the cell culture medium on the electrochemical quantification of H2O2. In this work, we studied the effect of different media (RPMI, MEM, DMEM, Ham’s F12 and BEGM/DMEM) on the electrochemical quantification of H2O2. The used electrode is based on reduced graphene oxide (rGO) and gold nanoparticles (AuNPs) and was obtained by co-electrodeposition. To reduce the electrode fouling by the medium, the effect of dilution was investigated using diluted (50% v/v in PBS) and undiluted media. With the same aim, two electrochemical techniques were employed, chronoamperometry (CH) and linear scan voltammetry (LSV). The influence of different interfering species and the effect of the operating temperature of 37 °C were also studied in order to simulate the operation of the sensor in the culture plate. The LSV technique made the sensor adaptable to undiluted media because the test time is short, compared with the CH technique, reducing the electrode fouling. The long-term stability of the sensors was also evaluated by testing different storage conditions. By storing the electrode at 4 °C, the sensor performance was not reduced for up to 21 days. The sensors were validated measuring H2O2 released by two different human bronchial epithelial cell lines (A549, 16HBE) and human primary bronchial epithelial cells (PBEC) grown in RPMI, MEM and BEGM/DMEM media. To confirm the results obtained with the sensor, the release of reactive oxygen species was also evaluated with a standard flow cytometry technique. The results obtained with the two techniques were very similar. Thus, the LSV technique permits using the proposed sensor for an effective oxidative stress quantification in different culture media and without dilution.

Formoterol Exerts Anti-Cancer Effects Modulating Oxidative Stress and Epithelial-Mesenchymal Transition Processes in Cigarette Smoke Extract Exposed Lung Adenocarcinoma Cells

“…CSE increases the extracellular ROS in adenocarcinoma cells, confirming the data obtained with flow cytometry. The quantification of H 2 O 2 , by using a specific electrochemical sensor only for H 2 O 2 , in cellular supernatants of A549 is a convenient, easy, and validated way to monitor the oxidative stress of the cell [27].…”

“…The extracellular oxidative stress was evaluated using an innovative electrochemical sensor that, as demonstrated [27], was able to detect hydrogen peroxide. The sensor was used to quantify hydrogen peroxide in cell supernatant collected from CSE-and/or FO-stimulated A549 cells.…”

Role of airway epithelium in viral respiratory infections: Can carbocysteine prevent or mitigate them?

“…Engineering studies have developed electrochemical sensors that can monitor oxidative stress in in vitro models and biological fluids in a non‐invasive, direct, cheap, easy, and validated way. In this way, the progression of the disease, and the response to the therapy can be followed [86]. The use of these sensors associated with advanced models offers valid support to pre‐clinical research (Figure 4).…”

Oxidative Stress, Environmental Pollution, and Lifestyle as Determinants of Asthma in Children

“…All this considered, ROS and RNS derivatives have been proposed as asthma biomarkers, especially regarding severe, neutrophilic asthma [4][5][6]89]. Although ROS are too reactive, unstable, and compartmentalized to be directly measured, in recent years, there has been a growing increase in studies carried out by multidisciplinary research groups for development of electrochemical sensors that enable measurement of some more stable ROS in cell line culture media with the aim of being able to apply them directly on patients [92,93]. Furthermore, many secondary products of their excess have been proposed as indirect redox markers in many different biological samples, such as urine, blood, bronchoalveolar lavage (BAL), induced sputum, or exhaled breath condensate [4,5,7,10,59,89,90,94].…”