A study of the chemiluminescence behavior of myoglobin with luminol and its analytical applications.

A chemiluminescence signal at 425 nm was observed when ferric state myoglobin was mixed with luminol in alkaline medium. Because the signal was remarkably enhanced in the presence of Fe(CN)6(4-), analytical applications were investigated in a flow-injection system. The increase in chemiluminescence was linearly dependent on myoglobin concentration in the range 0.1 to 100 nmol L(-1), and the limit of detection was 0.04 nmol L(-1) with relative standard deviation 3.2% (3 sigma). It was also found that binding of Mb with the ligands CN-, SCN-, and F- significantly inhibited the chemiluminescence reaction. The linear dynamic ranges for the ligands were 1.0-300.0, 0.1-3.0, and 0.5-100.0 nmol L(-1), and the limits of detection (S/N=3) 0.4, 0.04, and 0.2 nmol L(-1), for F-, CN-, and SCN-, respectively. The relative standard deviations were 5.32%, 6.13%, and 3.38% for 0.1 nmol L(-1) CN-, 0.5 nmol L(-1) SCN-, and 1.0 nmol L(-1) F-, respectively. At a flow rate of 2.0 mL min(-1) the assay could be accomplished in 1 min, including sampling and washing. The method has been successfully applied to the determination of myoglobin in human urine and F- in water samples. A possible mechanism of chemiluminescence production by myoglobin and luminol is presented.