New method for the determination of extracellular production of superoxide by marine phytoplankton using the chemiluminescence probes MCLA and red-CLA

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 superoxidespecific 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 µM) 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 redCLA, 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