Reactive Species Detection in Biology || UV–Vis Absorption and Chemiluminescence Techniques

摘要： Spectrophotometric techniques involving ultraviolet and colorimetric detection offer a convenient way of detecting reactive species (RS) formation due to the prevalence of UV-Vis spectrophotometer in research laboratories. Conventional spectrophotometric measurement of RS exploits their unique chemical reactivity with a small organic molecule and/or enzyme that has specificity to these RS where formation (or disappearance) of absorption peak/s at a particular wavelength is observed at the UV to the visible region of the electromagnetic spectrum. Direct detection of O2 from xanthine oxidase in the past involves rapid-freeze technique using electron paramagnetic resonance (EPR) spectroscopy. At the same time, spectrophotometric detection of O2 generated from xanthine oxidase/xanthine system was also employed for the investigation of the enzymatic property of superoxide dismutase (SOD) where the O2 levels were measured through reduction of ferricytochrome c, tetranitromethane, or oxidation of epinephrine to adenochrome. Several approaches have been developed since then to improve sensitivity with the use of submicromolar probe concentration thus allowing minimal interferences of the probe on the biological process being investigated. Specificity has also been improved to increase the reaction rate of probes to certain RS through synthesis of new and innovative analogues that exploit the unique chemistry between the probe and RS. Chromophore stability was also achieved through improved molecular design, optimized experimental conditions, or addition of supramolecular reagents since chromophores impart inherent thermodynamic stability as a function of its chemical structure, solvent polarity, pH, temperature, or due to presence of oxido-reductants or other reactive substances. Improved sample preparation and high-throughput analysis were also developed in order to maximize efficiency in the measurement markers of oxidative stress and determination of antioxidant capacity (AOC) of known molecules, food, biological fluid, or tissue. Therefore, spectrophotometric techniques for RS measurement have found broad application in the fields of biomedical research, clinical chemistry, plant biology, food chemistry, environmental chemistry, radiation chemistry, pharmaceuticals, toxicology, or material science to name a few, or just simply for the investigation of RS production in simple chemical systems. However, unlike the fluorescence probes, spectrophotometric as well as most applications of chemiluminescence probes do not provide spectrospatial image of the RS localization in cells, hence, one cannot deduce the site of radical production unless multiple probes and/or inhibitors are used with varying compartmentalization property (i.e., extracellular or intracellular). For example, in the investigation of radical production in cellular NADPH oxidase, several methods had been suggested such as measurement of O2 consumption, use of SOD-ferricytochrome c and horseradish peroxidase (HRP)/inhibitable probe such as Amplex Red for extracellular O2 and H2O2, respectively, and the HPLC analysis of the 2-OH-E marker for the quantification of intracellular O2.