Bimetallic Single-Atom Nanozyme-Based Electrochemical-Photothermal Dual-Function Portable Immunoassay with Smartphone Imaging

Rapid and accurate detection of human epidermal growth factor receptor 2 (HER2) is crucial for the early diagnosis and prognosis of breast cancer. In this study, we reported an iron–manganese ion N-doped carbon single-atom catalyst (FeMn-NC_etch/SAC) bimetallic peroxidase mimetic enzyme with abundant active sites etched by H₂O₂ and further demonstrated unique advantages of single-atom bimetallic nanozymes in generating hydroxyl radicals by density functional theory (DFT) calculations. As a proof of concept, a portable device-dependent electrochemical-photothermal bifunctional immunoassay detection platform was designed to achieve reliable detection of HER2. In the enzyme-linked reaction, H₂O₂ was generated by substrate catalysis via secondary antibody-labeled glucose oxidase (GOx), while FeMn-NC_etch/SAC nanozymes catalyzed the decomposition of H₂O₂ to form OH*, which catalyzed the conversion of 3,3′,5,5′-tetramethylbenzidine (TMB) to ox-TMB. The ox-TMB generation was converted from the colorimetric signals to electrical and photothermal signals by applied potential and laser irradiation, which could be employed for the quantitative detection of HER2. With the help of this bifunctional detection technology, HER2 was accurately detected in two ways: photothermally, with a linear scope of 0.01 to 2.0 ng mL^–1 and a limit of detection (LOD) of 7.5 pg mL^–1, and electrochemically, with a linear scope of 0.01 to 10 ng mL^–1 at an LOD of 3.9 pg mL^–1. By successfully avoiding environmental impacts, the bifunctional-based immunosensing strategy offers strong support for accurate clinical detection.