Micelle enhanced spectrofluorimetric assay for laccase activity by a flow-injection stopped-flow technique

An automated flow-injection stopped-flow kinetic spectrofluorimetric method for determination of laccase activity is proposed. It is based on the oxidation of o-phenylenediamine (1,2-diaminobenzene) catalysed by laccase during the stopped-flow period, yielding 2,3-diaminophenazine, which is determined by a micelle enhanced spectrofluorimetric method in a non-ionic surfactant medium. The substrate solution with Brij-35 and the buffer with sample are pumped into the flow manifold at the same flow rate and merged in the flow cell, then stopped for measuring the fluorescence intensity (λex = 425 nm, λem = 530 nm) which increases with time, at a constant temperature of 46°C. The linear range of the method is 0.1–118 U l−1 with a detection limit of 0.07 U l−1, using a 5 min stopped-flow reaction time. The proposed method has been applied to the assay for laccase activity of real samples at 10 samples per h.