Simple hydrated electron-induced electrochemiluminescence determination of alkaline phosphatase with novel time-resolved internal standard strategy at boron doped diamond electrodes

Hydrated electrons were generated at boron doped diamond electrodes in fully aqueous solutions during cathodic pulse polarization and utilized for the electrochemiluminescence (ECL) determination of alkaline phosphatase (ALP) by adapting one of the most common fluorescent methods. In this approach, highly fluorescent 4-methylumbelliferyl (MU) is enzymatically generated from non-fluorescent 4-methylumbelliferyl phosphate (MUP) substrate and chemically excited in the presence of oxidizing radicals and reducing hydrated electrons. A novel time-resolved internal standard strategy is presented to counter matrix effects to certain degree and to lower signal variation among concurrent measurements. In this unique approach, the cathodic ECL signals of MU and Tb(III)-chelate were separated on the basis of their exceptionally different excited state lifetimes. This enabled more precise determination of ALP by simply measuring the ratio of ECL emissions during and after cathodic excitation pulse with simple two-electrode, single photomultiplier tube setup. The ratiometric ECL signals in the absence and presence of 1.0 % serum (v/v) were interchangeable whereas ratiometric signal recovery in the presence of 10.0 % serum was similar to 85 %.