In Situ SERS Detection of Hydrogen Peroxide Released from Cells using Ag@Au Core/Shell Nanoparticle Nanomembrane as Substrate

Hydrogen peroxide (H₂O₂) is a key reactive oxygen species (ROS) that regulates redox signals in organisms by reversibly oxidizing target proteins. However, it causes oxidative damage at supraphysiological concentrations, leading to various diseases. Therefore, accurate and sensitive detection of H₂O₂ in organisms, especially in cells, is essential for reliable pathological diagnosis. Herein, a sensitive and reproducible surface-enhanced Raman spectroscopy (SERS) platform was developed for in situ profiling detection of H₂O₂ released from cells utilizing a uniform and dense plasmonic Ag@Au core/shell nanoparticle nanomembrane (Ag@AuNPs-NM) as the SERS substrate. In the designed substrate configuration, the Ag@Au core/shell nanostructure combines the strong electromagnetic field enhancement of AgNPs with the stability of AuNPs, which could enhance the sensitivity of SERS detection. Meanwhile, the dense and uniform distribution of "hot spots" in the Ag@AuNPs-NM endows the platform with excellent detection reproducibility. In the presence of H₂O₂, the ratiometric I₁₀₇₃/I₉₉₈ SERS signal of the Raman reporter 4-mercaptophenylboronic acid (4-MPBA) is changed due to the oxidation of the boronate group of 4-MPBA to the phenolic hydroxyl group of 4-hydroxythiophenol (4-HTP). The developed SERS sensing platform possesses a good linear relationship between the ratiometric I₁₀₇₃/I₉₉₈ signal of 4-MPBA and the concentration of H₂O₂ in the range from 1 to 500 μM, with a limit of detection of 0.26 μM. As a proof of concept, we applied this platform to in situ monitoring of drug-stimulated H₂O₂ released from cells that were cultured on the SERS chips. This work provides a practical method for the in situ profiling detection of cellular H₂O₂ with satisfactory results, holding promise for the diagnosis of H₂O₂-associated diseases.