Optimization of peroxynitrite-luminol chemiluminescence system for detecting peroxynitrite in cell culture solution exposed to carbon disulphide.

We established a peroxynitrite-luminol chemiluminescence system for detecting peroxynitrite in cell culture solution exposed to carbon disulphide (CS(2)). Three factors, including exposure time to ozone (Factor A), volume of peroxynitrite (ONOO(-)) solution (Factor B) and luminol concentrations (Factor C) at three levels were selected and the combinations were in accordance with orthogonal design L(9) (3(4)). Peroxynitrite was generated from the reaction of ozone and 0.01 mol/L sodium azide (NaN(3)) dissolved in carbonic acid buffer solution (pH 11), and it was reacted with luminol to yield chemiluminescence. The peak value, peak time and kinetic curve of the light emission were observed. The selected combination conditions were 50 s ozone, 800 micro L peroxynitrite and 0.001 mol/L luminol solution. Cell culture solution with CS(2) enhanced the emission intensity of chemiluminescence (F = 8.38, p = 0.018) and shortened the peak time to chemiluminescence (F = 139.00, p = 0.0001). The data demonstrated that this luminol chemiluminescence system is suitable for detecting peroxynitrite in cell culture solutions for evaluating the effect of CS(2) on endothelial cells.