New method for the determination of extracellular production of superoxide by marine phytoplankton using the chemiluminescence probes MCLA and red-CLA
New method for the determination of extracellular production of superoxide by marine phytoplankton using the chemiluminescence probes MCLA and red-CLA
复制标题
DOI：
10.4319/lom.2009.7.682
复制
发表时间：
2009-10-01
影响因子：
2.7
通讯作者：
Waite, T. David
中科院分区：
文献类型：
--
作者：
Godrant, Aurelie;Rose, Andrew L.;Waite, T. David
建立了一种快速、高灵敏度的微板化学发光法测定浮游植物胞外超氧化物的新方法。将样品、空白和三种标准品的重复样品置于96孔板中，并用微孔板光度计检测化学发光。使用两种超氧化物特异性荧光探针，MCLA和密切相关的化合物红-CLA，分别在460和610 nm处发光，在超氧化物的存在下，该方法进行了测试Trichodesmium alumraeum文化。通过加入固定浓度的黄嘌呤（50 μ M）和可变浓度的黄嘌呤氧化酶（0.001-0.5单位L-1），使用黄嘌呤/黄嘌呤氧化酶系统对每个样品单独进行荧光信号的校准。该方法对超氧阴离子具有选择性，检测限为1.41 pmol/s（MCLA）和76 fmol/s（red-CLA），定量限为4.70 pmol/s（MCLA）和253 fmol/s（red-CLA）。新方法的应用，以确定由多产的超氧化物产生的浮游植物Chattonella marina的胞外超氧化物产生的结果相比，使用现有的流动注射分析方法。与现有方法相比，使用微孔板具有几个优点，包括空白、样品和标准品一式三份的分析时间短（10 min）;信号可靠性好;以及使用小样品体积。
A new rapid and highly sensitive microplate-based chemiluminescence method for the detection of extracellular production of superoxide by phytoplankton cultures has been developed. Replicates of the sample, blank, and three standards were placed into 96-well plates and the chemiluminescence was detected with a microplate luminometer. The method was tested on Trichodesmium erythraeum cultures using two superoxide-specific chemiluminescent probes, MCLA and the closely related compound red-CLA, which emit light at 460 and 610 nm, respectively, in the presence of superoxide. Calibration of the chemiluminescent signal is performed individually for each sample using the xanthine/xanthine oxidase system by adding a fixed concentration of xanthine (50 mu M) and variable concentrations of xanthine oxidase (0.001-0.5 units L-1). The method is selective for superoxide, and the detection limits are as low as 1.41 pmol/s for MCLA and 76 fmol s(-1) for red-CLA, with limits of quantification of 4.70 pmol/s for MCLA and 253 fmol s(-1) for red-CLA. Application of the new method to the determination of extracellular superoxide production by the prolific superoxide-producing phytoplankton Chattonella marina yielded results comparable to those obtained using an existing flow injection analysis method. The use of microplates offers several advantages over existing methods, including a short analysis time of 10 min for triplicates of blank, sample, and standards; good reliability of signals; and use of small sample volumes.