Abstract
o-Phenylenediamine (OPD) is commonly used as a reliable signaling agent for colorimetric assays via oxidative dimerization to 2,3-diaminophenazine (DAP) (λmax = 425 nm), which is catalyzed by conventional horseradish peroxidase (HRP) or its nanoparticle mimics. Recently, it has been reported that catalytic and electrochemical oxidation of OPD produces a mixture of polymerized OPD molecules (polyOPDs), which could potentially affect the colorimetric signal due to the difference in optical properties between DAP and polyOPDs. In our study, we present for the first time that the gold nanoparticle-catalyzed oxidation of OPD could exhibit nonmonotonic extinction transitions at 425 nm. Using various spectroscopic and microscopic techniques such as UV-vis spectroscopy, transmission electron microscopy, dynamic light scattering (DLS), and Fourier transform infrared (FT-IR) spectroscopy, we verify that the production of polyOPDs is specifically responsible for the unexpected decrease in extinction at 425 nm. This discovery presents a potential challenge to the conventionally accepted role of OPD as a signaling agent. Furthermore, we find that the modification of reaction variables, including reactant concentrations, anion types, and temperature, determines how nonmonotonic the extinction transition could be. Lastly, we develop an OPD-based colorimetric DNA detection scheme using DNA-functionalized gold nanoparticles to demonstrate the potential problems in accurately quantifying the target. Our proposal of using NaNO3 instead of NaCl to provide the desired ionic strength could be a suitable solution to overcome the obstacles of detection.