Graduate School of Biosphere Science, Department of Environmental Dynamics and Management, Hiroshima University , 1-7-1 Kagamiyama, Higashi-Hiroshima, Hiroshima 739-8521, Japan.
Anal Chem. 2015 Dec 15;87(24):11998-2005. doi: 10.1021/acs.analchem.5b00917. Epub 2015 Nov 24.
Superoxide radical (O2(•-)) is an important reactive oxygen species in seawater. Measurements of its production rates and steady-state concentrations generated by photochemical processes have been a Herculean task over the years. In this study, a probe - 3'6'-(diphenylphosphinyl)fluorescein (PF-1) - was used to trap photochemically generated O2(•-) in seawater, thereby yielding fluorescein. The fluorescein produced was measured by an isocratic fluorescence HPLC at excitation/emission wavelengths of 490/513 nm, respectively. The reaction rate constant of PF-1 with O2(•-) (kPF-1) was pH-dependent: (3.2-23.5) × 10(7) M(-1) s(-1) at pHTOT 7.65-8.50. By applying appropriate equations, both the production rate and the steady-state concentration of O2(•-) generated by photochemical reactions in the seawater were quantified. Under the optimized experimental conditions, fluorescein standards (3-50 nM) exhibited linearity in the seawater by HPLC. The photoformation of fluorescein, due to the reaction of PF-1 with the O2(•-) photochemically produced in the seawater, was linear within the 20 min irradiation. The detection limit of the fluorescein photoformation rate was 0.03 pM s(-1), defined as 3σ of the lowest standard fluorescein concentration per 20 min irradiation. Using this value, the yield of fluorescein, and the fraction of O2(•-) that reacted with PF-1 in the seawater, the detection limit of the O2(•-) photoformation rate was 1.78 pM s(-1). Superoxide measurements using the proposed method were relatively unaffected by the potential interfering species in seawater. Application of the proposed method to ten (10) seawater samples from the Seto Inland Sea, Japan, resulted in measured O2(•-) photoformation rates of 3.1-8.5 nM s(-1), with steady-state concentrations ranging (0.06-0.3) × 10(-10) M. The method is simple, requires no technical sample preparation, and can be used to analyze a large number of samples.
超氧自由基 (O2(•-)) 是海水中一种重要的活性氧物质。多年来，测量其光化学反应产生的生成速率和稳态浓度一直是一项艰巨的任务。在本研究中，使用探针 3'6'-(二苯基膦酰基)荧光素 (PF-1) 来捕获海水中光化学反应产生的 O2(•-)，从而生成荧光素。生成的荧光素通过等度荧光 HPLC 在激发/发射波长分别为 490/513nm 下进行测量。PF-1 与 O2(•-) 的反应速率常数 (kPF-1) 随 pH 变化：在 pHTOT 7.65-8.50 时为 (3.2-23.5) × 10(7) M(-1) s(-1)。通过应用适当的方程，定量了海水中光化学反应生成的 O2(•-) 的生成速率和稳态浓度。在优化的实验条件下，HPLC 中海水中的荧光素标准品 (3-50nM) 表现出线性关系。由于 PF-1 与海水中光化学产生的 O2(•-) 反应，荧光素的光形成在 20 分钟的辐照内呈线性关系。荧光素光形成速率的检测限为 0.03 pM s(-1)，定义为每 20 分钟辐照最低标准荧光素浓度的 3σ。使用该值、荧光素的产率以及海水中与 PF-1 反应的 O2(•-) 分数，可以得出 O2(•-) 光形成速率的检测限为 1.78 pM s(-1)。使用所提出的方法测量超氧自由基时，海水潜在干扰物质的影响相对较小。将所提出的方法应用于来自日本濑户内海的 10 个海水样本，得到的 O2(•-) 光形成速率为 3.1-8.5nM s(-1)，稳态浓度范围为 (0.06-0.3) × 10(-10) M。该方法简单，无需技术样品制备，可用于分析大量样品。
