Engineering Two-Dimensional-Copper Phenolic Nanosheet from CuO Nanosphere for Enhanced Peroxidase Activity in Smartphone-Based Thiophanate-Methyl Detection via Analyte-Inhibition Mechanism

Abstract

Optical sensors based on nanozymes have shown great promise for the rapid and sensitive detection of pesticide residues in environmental and food samples. However, their applicability is often limited by a lack of selectivity due to nonspecific enzyme inhibition. Therefore, we developed a colorimetric sensing platform specifically tailored to detect the agricultural fungicide thiophanate-methyl (TpM), leveraging its unique inhibitory effect on nanozyme activity. The symmetric ethylenediamine- and bisthiourea-like groups in TpM exhibit a strong affinity for metal sites, leading to a reduction in the catalytic performance. Thus, we designed a novel copper oxide nanosphere (CuO)-derived 2D copper phenolic nanosheet (2D-CuPNS) with enhanced peroxidase-like activity. This nanosheet facilitates the oxidation of TMB through OH radical generation via a Fenton-like reaction involving Cu²⁺/Cu⁺ and −C═C– bonds on the nanozyme surface. Notably, the activity of 2D-CuPNS was selectively and significantly inhibited by TpM due to a specific interaction while remaining unaffected by other pesticides. The 2D-CuPNS exhibited strong affinities for TMB and H₂O₂, with K_m values of 0.07 and 2.0 mM, respectively. As a result, the proposed sensor demonstrated high specificity for TpM detection, with a linear range of 0.5–15 μM and a low detection limit of 0.09 μM. A smartphone-assisted rapid detection method was also developed utilizing RGB values from the chromogenic reaction. Furthermore, by immobilization of the 2D-CuPNS and TMB substrate onto standard filter paper, ready-to-use paper-based TpM sensors were created. This study introduces a novel nanozyme inhibition approach for the selective detection of TpM in environmental and food samples, providing a versatile and practical sensing solution.