Department of Chemistry and Biochemistry, University of Delaware, Newark, DE, 19711, USA.
Anal Bioanal Chem. 2019 Aug;411(20):5287-5296. doi: 10.1007/s00216-019-01910-2. Epub 2019 Jun 15.
Singlet oxygen (O) is the focus of study in many fields, including phototoxicity, antioxidant activity, pollutant weathering, photodynamic therapy, and water disinfection. The imidazole plus RNO (Imd/RNO) method, originated by Kraljic and El Mohsni, is commonly used to monitor singlet oxygen production. In this method, O is quenched by an acceptor, imidazole (Imd), during the formation of a trans-annular peroxide intermediate that bleaches the sensor, p-nitrosodimethylaniline (RNO). Though the method has been widely used, including to monitor O production in complex environments, such as surfactants and cells, studies reporting the efficiency of the assay in complex solvents have not been reported. In this research, the Imd/RNO method in complex, biorelevant solvents, i.e., sodium dodecyl sulfate, octanol, and phosphate buffer-saturated octanol, was compared with reference solvents, i.e., phosphate buffer, ethanol, and methanol, for monitoring O produced by Rose Bengal photosensitization using time-resolved, broadband UV-Vis absorbance measurements. Rates of sensor bleaching and sensitizer photodegradation were simultaneously monitored in each solvent to investigate correlations between the disappearance rates of sensor and sensitizer. The quantum yields of O production (ϕ) in each solvent were calculated using a relative actinometric method. The dependence of sensor bleaching and sensitizer degradation on acceptor concentration and solvent polarity, and the results of assay controls suggest mechanistic differences underlying the reactions comprising the Imd/RNO method. These results demonstrate the need for caution and controls when using the method in complex samples including those containing cells, tissues, or nanoscale particles.
单线态氧 (O) 是许多领域研究的焦点，包括光毒性、抗氧化活性、污染物风化、光动力疗法和水消毒。Kraljic 和 El Mohsni 首创的咪唑加 RNO（Imd/RNO）方法常用于监测单线态氧的产生。在该方法中，O 在形成环间过氧化物中间体时被受体咪唑（Imd）猝灭，该中间体使传感器 p-亚硝二甲苯胺（RNO）漂白。尽管该方法已被广泛应用，包括监测复杂环境（如表面活性剂和细胞）中的 O 产生，但尚未有报道报道该测定方法在复杂溶剂中的效率。在这项研究中，在复杂的生物相关溶剂（如十二烷基硫酸钠、辛醇和磷酸盐缓冲液饱和辛醇）中比较了 Imd/RNO 方法与参考溶剂（如磷酸盐缓冲液、乙醇和甲醇），用于监测 Rose Bengal 敏化产生的 O 使用时间分辨宽带紫外-可见吸收测量。在每种溶剂中同时监测传感器漂白和敏化剂光降解的速率，以研究传感器和敏化剂消失速率之间的相关性。使用相对放射性测量法计算了每种溶剂中 O 产生的量子产率（ϕ）。传感器漂白和敏化剂降解对受体浓度和溶剂极性的依赖性以及测定对照的结果表明，Imd/RNO 方法中包含的反应具有不同的机制。这些结果表明，在含有细胞、组织或纳米级颗粒等复杂样品中使用该方法时需要谨慎和控制。
