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 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.