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Hydroxyl radicals (•OH) are known as essential chemicals for cells to maintain their normal functions and defensive responses. However, a high concentration of •OH may cause oxidative stress-related diseases, such as cancer, inflammation, and cardiovascular disorders. Therefore, •OH can be used as a biomarker to detect the onset of these disorders at an early stage. Reduced glutathione (GSH), a well-known tripeptide for its antioxidant capacity against reactive oxygen species (ROS), was immobilized on a screen-printed carbon electrode (SPCE) to develop a real-time detection sensor with a high selectivity towards •OH. The signals produced by the interaction of the GSH-modified sensor and •OH were characterized using cyclic voltammetry (CV) and electrochemical impedance spectroscopy (EIS). The CV curve of the GSH-modified sensor in the Fenton reagent exhibited a pair of well-defined peaks, demonstrating the redox reaction of the electrochemical sensor and •OH. The sensor showed a linear relationship between the redox response and the concentration of •OH with a limit of detection (LOD) of 49 µM. Furthermore, using EIS studies, the proposed sensor demonstrated the capability of differentiating •OH from hydrogen peroxide (H2O2), a similar oxidizing chemical. After being immersed in the Fenton solution for 1 hr, redox peaks in the CV curve of the GSH-modified electrode disappeared, revealing that the immobilized GSH on the electrode was oxidized and turned to glutathione disulfide (GSSG). However, it was demonstrated that the oxidized GSH surface could be reversed back to the reduced state by reacting with a solution of glutathione reductase (GR) and nicotinamide adenine dinucleotide phosphate (NADPH), and possibly reused for •OH detection. 

Reactive oxygen species (ROS) have been found in plants, mammals, and natural environmental processes. The presence of ROS in mammals has been linked to the development of severe diseases, such as diabetes, cancer, tumors, and several neurodegenerative conditions. The most common ROS involved in human health are superoxide (O2•−), hydrogen peroxide (H2O2), and hydroxyl radicals (•OH). Organic and inorganic molecules have been integrated with various methods to detect and monitor ROS for understanding the effect of their presence and concentration on diseases caused by oxidative stress. Among several techniques, fluorescence and electrochemical methods have been recently developed and employed for the detection of ROS. This literature review intends to critically discuss the development of these techniques to date, as well as their application for in vitro and in vivo ROS detection regarding free-radical-related diseases. Moreover, important insights into and further steps for using fluorescence and electrochemical methods in the detection of ROS are presented. 

It is well known that an excess of hydroxyl radicals (˙OH) in the human body is responsible for oxidative stress-related diseases. An understanding of the relationship between the concentration of ˙OH and those diseases could contribute to better diagnosis and prevention. Here we present a supersensitive nanosensor integrated with an electrochemical method to measure the concentration of ˙OH in vitro. The electrochemical sensor consists of a composite comprised of ultrasmall cerium oxide nanoclusters (<2 nm) grafted to a highly conductive carbon deposited on a screen-printed carbon electrode (SPCE). Cyclic voltammetry (CV) and electrochemical impedance spectroscopy (EIS) were used to analyze the interaction between cerium oxide nanoclusters and ˙OH. The CV results demonstrated that this electrochemical sensor had the capacity of detecting ˙OH with a high degree of accuracy and selectivity, achieving a consistent performance. Additionally, EIS results confirmed that our electrochemical sensor was able to differentiate ˙OH from hydrogen peroxide (H 2 O 2 ), which is another common reactive oxygen species (ROS) found in the human body. The limit of detection (LOD) observed with this electrochemical sensor was of 0.6 μM. Furthermore, this nanosized cerium oxide-based electrochemical sensor successfully detected in vitro the presence of ˙OH in preosteoblast cells from newborn mouse bone tissue. The supersensitive electrochemical sensor is expected to be beneficially used in multiple applications, including medical diagnosis, fuel–cell technology, and food and cosmetic industries.