Mechanistic insights into the competitive inhibition of enzyme-mimetic activity of gold nanoparticles for dual mode colorimetric and fluorescence detection of biothiols

In this work, the interaction behaviour of gold nanoparticles (AuNPs) with o-phenylenediamine (OPD) was studied to ascertain the nanozyme-substrate interaction. The UV-Vis absorption, high-resolution transmission electron microscopy and zeta potential analysis revealed that the electron-rich nitrogen atoms in OPD showed a stronger affinity toward electron-deficient surface, indicating a stronger interaction between nanozyme and substrate molecules. Subsequently, under optimum conditions, AuNPs are used as nanozyme to catalyze the oxidation of OPD in the presence of H₂O₂. The catalyzed product (2,3-diaminophenazine, (DAP)) generated visible colorimetric readout (yellow color) and showed yellow fluorescence upon excitation at 450 nm. The nanozyme-based oxidation reaction of OPD was then applied to detect glutathione (GSH) by colorimetric and fluorometric techniques. The detection principle is based on the fact that GSH being a thiol-containing moiety can readily interact with AuNPs and considerably decrease the catalytic activity of nanoparticles. In the presence of varying concentrations (1-15 µM) of GSH, the formation of DAP is significantly decreased leading to a decrease in the absorbance and fluorescence intensity at 450 nm and 540 nm, respectively. The colorimetric and fluorescence assay for GSH exhibited a limit of detection of 3.42 and 2.01 µM, respectively. Kinetic studies were conducted to elucidate the inhibition mechanism of GSH on the catalytic function of AuNPs. To demonstrate the practical applicability of the nanozyme-based assay, GSH detection in artificial urine samples were carried out.