Colorimetric multiplexed immunoassay using specific aggregation of antigenic peptide-modified luminous nanoparticles

Two amperometric enzyme biosensor systems, based on glycerol dehydrogenase/diaphorase (GDH/DP) and glycerol kinase/glycerol-3-phosphate oxidase/peroxidase (GK/GPOx/HRP), were developed and used for estimation of glycerol content in a complex biological fluids. Enzymes were immobilized on interchangeable membranes by PCS-prepolymer technique. Buffers containing ferricyanide/NAD⁺ or ferrocyanide/ATP were used for measurements with GDH/DP and GK/GPOx/HRP biosensor, respectively. FIA assay of glycerol biosensor was characterized by a linear range of 0.01-1 or 0.01-1.5 mM glycerol, sensitivity of 6.02 or 1.42 mA/M cm² and with signal loss of 40% after 90 h or 30% after 16 h during continuous operation at a sample throughput of 10 injections/h for GDH/DP or GK/GPOx/HRP biosensors, respectively. Both biosensors were successfully used for off-line monitoring of glycerol during microbial transformation of glycerol to 1,3-propanediol using an automatized flow-through system. The results were consistent with those obtained with HPLC. The stability of described biosensor systems was sufficient for monitoring and control of fermentation process within 24 h. The storage stability of enzyme membranes was several months.