Towards mechanized biparametric ceruloplasmin assay.

Two analytical strategies for determination of both biocatalytic activity and concentration of ceruloplasmin conditions have been proposed. For this purpose, two constructions of fully-mechanized Multicommutated Flow Analysis (MCFA) systems were designed. The versatility of solenoid pumps and valves arrangement enabled to construct both manifolds using similar flow units, taking into account the different requirements for each method. In the case of ceruloplasmin catalytic activity assay, the kinetic measurements with the use of p-phenylenediamine and hydrogen peroxide were performed. The optimization process was focused on the selection of substrate and oxidizer concentration, incubation time as well as solving the issue of substrate autoxidation. It led to the development of the flow bioanalytical system characterized by following analytical parameters: LOD - 0.07 U mL- 1, LOQ - 0.38 U mL-1, RSD ≤6% with 8 μL consumption of human serum. In turn, for examination of ceruloplasmin concentration, the light-scattering detector was used in MCFA system adapted for immunoprecipitation measurements. In this case, the use of potentiator (polyethylene glycol) turned out to be necessary to obtain satisfactory analytical signals. Such a method allowed to obtain 35 measurements per hour with LOD and LOQ of 0.9 mg L-1 and 3.2 mg L-1, respectively. The usefulness of both MCFA systems was successfully examined by performing analyses of real human serum samples.