Unravelling the Multi-Enzymatic Activity of Platinum Nanoparticles

Nanozymes are a new class of nanomaterials that can mimic the activity of some of the natural enzymes. [1] Thanks to their versatility, robustness, low manufacturing costs, and long shelf life, which make them easier to handle compared to their natural counterparts, nanozymes have been widely applied in clinical diagnostic, wastewater pollutant remediation, and have been proposed as antimicrobial, environmental treatments, anticancer agents, etc. [2] Although the effect of the nanozymes’ activity on the substrates is similar to natural enzymes, their catalytic mechanisms are drastically different, and usually suffer for lower specificity and lower activity. [3] These aspects are often overlooked in literature, to the point that nanomaterials that have dominated the field in the last decade for their oxidase- (OX) and peroxidase-like (POD) activities, such as cerium and iron oxide nanoparticles, have recently been questioned as catalysts due to their very low activity and efficiency. [4], [5] Thus, studies aiming to clarify the real advantages and drawbacks of nanozymes in respect to their natural counterparts are strongly demanded in the field, especially for new and emerging nanomaterials.Recently, some nanozymes possessing multi-enzymatic activity have emerged. [6] Among them, platinum nanoparticles (PtNPs), displaying OX, POD, and catalase-like (CAT) activities, are gaining momentum, especially in the field of nanomedicine for reactive oxygen species scavenging, [7] antioxidant detection, [8] and antimicrobial. [9] Nevertheless, there is still much to disclose concerning the PtNPs enzyme-like activity mechanisms and how these are affected by environmental parameters. Most of the studies on the topic are based on computational calculations, with little, if any, [10] experimental validation, and often performed on alloys rather than pure platinum nanoparticles. [11] The lack of in-depth knowledge in this regard hinders the development of new PtNP applications and the current understanding of the PtNP biological behavior.In this work, we performed an in-depth systematic study investigating the three known oxidoreductase-like activities of the PtNPs as a function of different environmental chemical and physical parameters, varying pH, temperature, buffer media, and substrates. We observed that PtNPs are generally more active at acidic pH, and that their activity increases with the temperature. Interestingly, CAT-like activity was found to be strongly dependent on the buffer composition, while such dependency was less marked for OX- and POD-like activities. Different colorimetric assays were commonly used in this field to test nanozymes’ activities. Surprisingly, we found that most of them can easily produce misleading results, due to the instability of the chromogenic probes in the different experimental conditions, or because the solvents, commonly employed to solubilize the substrates, even in commercial kits, interfere with the PtNPs activity. Furthermore, a sort of substrate selectivity of the nanozyme was observed, which was related to the affinity of the substrate for the surface of the PtNPs.In the second part of the study, we compared the Pt nanozymes with three natural enzymes: a mammal catalase for the CAT-like activity, a fungal laccase for the OX-like activity, and a horseradish peroxidase (HRP) for the POD-like activity. To compare the activity of PtNPs with its natural counterparts, we calculated their turnover frequency (TOF) in the same conditions, while, to compare the recyclability of the catalysts, we calculated the turnover number (TON). Despite the differences in their catalytic mechanisms, we found that the activity of PtNPs is generally comparable to the one of the natural enzymes and even higher in harsh conditions, where, on the contrary, their natural counterparts become inactive.In the last part of the study, we investigated the mechanisms underlying the PtNPs catalytic properties. Utilizing detection reagents with high selectivity for specific radicals, we determined that hydroxyl radical is the main player for CAT- and POD-like activities, while OX-like activity is strongly dependent on dissolved oxygen concentration. The three different enzyme-like activities are actually deeply interconnected and, although they occur simultaneously, one can be favored over the others tuning the relative concentration of reagents and catalyst.In summary, the present study provides a comprehensive characterization of PtNPs multi-enzyme activities and important insights on their catalytic mechanisms, which are of pivotal interest for the implementation of Pt-based nanozymes in sensing and nanomedicine applications.