Modulation of Alkaline Phosphatase Based ELISA in the Presence of Ions and Citrate Stabilized Nanoparticles

Alkaline phosphatase (ALP) is extensively used in various diagnostic assays including ELISA. Diagnostic methods/biosensors having a high limit of detection are often modified by the incorporation of nanoparticles. Nanoparticles are also known to enhance enzymatic activity. The aim of this study was to determine the effect of ions and nanoparticles of gold and silver on the catalysis of ALP and hence on the detection potential of ELISA. The nanoparticles were synthesized using citrate as a stabilizing and reducing agent. Both nanoparticles and ions bound ALP conjugated to goat anti-rabbit IgG as shown by UV–visible and fluorescence spectroscopic analysis. Maximum detection signal was achieved when ELISA was conducted in the presence of a combination of 0.8 µg/ml cit-AuNPs and 700 nM AgCl. The increase was by 2.65 and 3.67 folds when the combination was introduced at the substrate step and at the secondary antibody step, respectively. Molecular docking revealed that citrate binds to specific residues close to the substrate binding site leading to catalytically favoured structural changes in the conformation of ALP. The study provides a promising approach to modulate enzyme-associated diagnostic and therapeutic processes using ions and nanoparticles.