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This project was aimed to identify the quenching chemistry of biologically important reactive oxygen and nitrogen species (ROS/RNS, including radicals), to show antioxidant action against reactive species through H‐atom and electron transfer reactions, and to evaluate the ROS/RNS scavenging activity of antioxidants with existing analytical methods while emphasizing the underlying chemical principles and advantages/disadvantages of these methods. In this report, we focused on the applications and impact of existing assays on potentiating future research and innovations to evolve better methods enabling a more comprehensive study of different aspects of antioxidants and to provide a vocabulary of terms related to antioxidants and scavengers for ROS/RNS. The main methods comprise the scavenging activity measurement of the hydroxyl radical (•OH), dioxide(•1–) (O2 •–: commonly known as the superoxide radical), dihydrogen dioxide (H2O2: commonly known as hydrogen peroxide), hydroxidochlorine (HOCl: commonly known as hypochlorous acid), dioxidooxidonitrate(1–) (ONOO−: commonly known as the peroxynitrite anion), and the peroxyl radical (ROO•). In spite of the diversity of methods, there is currently a great need to evaluate the scavenging activity of antioxidant compounds in vivo and in vitro. In addition, there are unsatisfactory methods frequently used, such as non-selective UV measurement of H2O2 scavenging, producing negative errors due to incomplete reaction of peroxide with flavonoids in the absence of transition metal ion catalysts. We also discussed the basic mechanisms of spectroscopic and electrochemical nanosensors for measuring ROS/RNS scavenging activity of antioxidants, together with leading trends and challenges and a wide range of applications. This project aids in the identification of reactive species and quantification of scavenging extents of antioxidants through various assays, makes the results comparable and more understandable, and brings a more rational basis to the evaluation of these assays and provides a critical evaluation of existing ROS/RNS scavenging assays to analytical, food chemical, and biomedical/clinical communities by emphasizing the need for developing more refined, rapid, simple, and low‐cost assays and thus opening the market for a wide range of analytical instruments, including reagent kits and sensors.

Since an unbalanced excess of reactive oxygen/nitrogen species (ROS/RNS) causes various diseases, determination of antioxidants that can counter oxidative stress is important in food and biological analyses. Optical/electrochemical nanosensors have attracted attention in antioxidant activity (AOA) assessment because of their increased sensitivity and selectivity. Optical sensors offer advantages such as low cost, flexibility, remote control, speed, miniaturization and on-site/in situ analysis. Electrochemical sensors using noble metal nanoparticles on modified electrodes better catalyze bioelectrochemical reactions. We summarize the design principles of colorimetric sensors and nanoprobes for food antioxidants (including electron-transfer based and ROS/RNS scavenging assays) and important milestones contributed by our laboratory. We present novel sensors and nanoprobes together with their mechanisms and analytical performances. Our colorimetric sensors for AOA measurement made use of cupric-neocuproine and ferric-phenanthroline complexes immobilized on a Nafion membrane. We recently designed an optical oxidant/antioxidant sensor using N,N-dimethyl-p-phenylene diamine (DMPD) as probe, from which ROS produced colored DMPD-quinone cationic radicals electrostatically retained on a Nafion membrane. The attenuation of initial color by antioxidants enabled indirect AOA estimation. The surface plasmon resonance absorption of silver nanoparticles as a result of enlargement of citrate-reduced seed particles by antioxidant addition enabled a linear response of AOA. We determined biothiols with Ellman reagent−derivatized gold nanoparticles.

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