Patella Bernardo, Vincenzo Serena Di, Zanca Claudio, Bollaci Luciano, Ferraro Maria, Giuffrè Maria Rita, Cipollina Chiara, Bruno Maria Giuseppina, Aiello Giuseppe, Russo Michele, Inguanta Rosalinda, Pace Elisabetta
Department of Engineering, University of Palermo, 90128 Palermo, Italy.
Institute of Traslational Pharmacology (IFT), National Research Council of Italy (CNR), 90146 Palermo, Italy.
Micromachines (Basel). 2022 Oct 18;13(10):1762. doi: 10.3390/mi13101762.
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 HO 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 HO. In this work, we studied the effect of different media (RPMI, MEM, DMEM, Ham's F12 and BEGM/DMEM) on the electrochemical quantification of HO. 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% / 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 HO 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.
氧化应激的量化是一项具有挑战性的任务，它有助于监测慢性炎症性呼吸道疾病。关于用于细胞培养基中过氧化氢（HO）电化学传感器的开发，文献中有不同的研究。然而，关于细胞培养基对HO电化学量化影响的数据非常有限。在这项工作中，我们研究了不同培养基（RPMI、MEM、DMEM、Ham's F12和BEGM/DMEM）对HO电化学量化的影响。所使用的电极基于还原氧化石墨烯（rGO）和金纳米颗粒（AuNPs），通过共电沉积获得。为了减少培养基对电极的污染，研究了使用稀释（50%/在PBS中）和未稀释培养基的稀释效果。出于同样的目的，采用了两种电化学技术，计时电流法（CH）和线性扫描伏安法（LSV）。还研究了不同干扰物质的影响以及37℃操作温度的影响，以模拟传感器在培养板中的操作。与CH技术相比，LSV技术使传感器能够适应未稀释的培养基，因为测试时间短，减少了电极污染。还通过测试不同的储存条件评估了传感器的长期稳定性。将电极储存在4℃下，传感器性能在长达21天内没有降低。通过测量在RPMI、MEM和BEGM/DMEM培养基中生长的两种不同人类支气管上皮细胞系（A549、16HBE）和人类原代支气管上皮细胞（PBEC）释放的HO，对传感器进行了验证。为了确认传感器获得的结果，还使用标准流式细胞术技术评估了活性氧的释放。两种技术获得的结果非常相似。因此，LSV技术允许使用所提出的传感器在不同培养基中且无需稀释地进行有效的氧化应激量化。
