The research of the catalysis mechanism and kinetics for peroxidase-like citrate capped gold nanoparticles on theoretical and experimental bases

The artificial enzymes, known as nanozymes, have wide range of applications by virtue of high stability against varying pH and temperature, low cost, preparation with desired size, shape and surface properties, high activity and unique electrochemical properties. Similar to some natural enzymes, they show activities such as peroxidase, oxidase, catalase and superoxide dismutase. Size, shape, surface modifications and net charge of nanozymes are critical to determine their enzyme-like activity. Those parameters can be tuned in order to design nanozymes with different levels of activity. The peroxidase-like activity of AuNPs make them attractive for researchers. Especially citrate-capped AuNPs have a wide range of applications. Although nanozymes such as AuNPs can be alternatives to natural enzymes, their structure and catalysis mechanisms could be different. In this study, the working principle of a nanozyme, citrate-capped AuNPs, was researched in detail and the catalysis mechanism was proposed to occur based on the adsorption of the substrates at numerous binding sites on the surface instead of substrates binding to a single active site as it happens with biological enzymes. Substrates of AuNPs are H2O2 and a chromogenic substrate, 3,3ʹ,5,5ʹ-tetramethylbenzidine (TMB). Different substrates can also be used instead of TMB. According to the proposed catalysis mechanism, both substrates adsorbed on the binding sites which are expected to be on AuNP surface. While H2O2 molecules are adsorb through hydrogen bonding, positively charged TMB molecules are adsorbed at negatively charged binding sites via electrostatic interactions. Adsorbed H2O2 molecules reveal hydroxyl radicals (•OH) as a result of a Fenton-like reaction, and the surface reaction in which TMB is oxidized takes place between •OH and adjacently adsorbed TMB as the rate-determining step. According to the explained surface reaction, in this study, Langmuir-Hinshelwood mechanism was proposed rather than commonly accepted Michaelis-Menten mechanism. Later, in order to elaborate the idea that the catalysis occurs for AuNPs through the adsorption at the binding sites all over the surface, DNA-modified citrate-capped AuNP and cysteamine-capped AuNP models were examined. It is hypothesized that the presence of DNA on particle surface improves adsorption by both increasing negative charge for TMB and providing new hydrojen bonding possibilities for H2O2. On the other hand, the amine groups on the surface of the cysteamine-capped particles mean less hydrogen bonding possibilities and positive charge in certain pH range, which results in reduced adsorption of both substrates. Hence, it was revealed that the presence of higher number of binding sites on the surface increases the amount of adsorbed substrates and thus the nanozyme activity for AuNPs. Similar relation can be observed as lowered total activity for smaller number of binding sites. So, the reasoning revealed that for a better explenation for nanozyme catalysis which includes surface reactions, Langmuir-Hinshelwood mechanism can be suggested rather than Michaelis-Menten mechanism which has been generally suggested in the literature for nanozymes. Following the mechanism discussion, mathematical modelling carried out for a comparison between Michaelis-Menten and Langmuir-Hinshelwood kinetics so that the correlation of both kinetic models with an experimental data set for citrate-capped AuNPs can be tested. As a result, a high correspondence both between two models and to the experimental data was observed. According to the results, it was deduced that Langmuir-Hinshelwood mechanism and kinetics can be utilized for citrate-capped AuNPs for a well-explained approach instead of Michaelis-Menten that has been rashly being used so far.