Nanozyme-SERS cascade immunoassay: From in-situ generation to signal enhancement of diffusible reporters in hotspots of Ag-rich nanoshell monolayers.

The ultrasensitive detection of protein biomarkers is critical for early disease diagnosis and intervention. Surface-enhanced Raman spectroscopy (SERS) offers high sensitivity and molecular specificity, yet conventional SERS immunoassays face two fundamental limitations: (i) in covalently labeled nanotags, the immunoprotein layers sterically separate the reporters from the electromagnetic hotspots of the SERS substrate, leading to signal attenuation; and (ii) electrostatically adsorbed reporters readily desorb and diffuse to the substrate during immunocomplex formation, generating false-positive signals. To address these limitations, we introduced a nanozyme-SERS cascade immunoassay that decouples signal generation from the immunocomplex. The platform employs Au@Ag-Pt nanozymes as catalytic probes and Ag-rich nanoshell monolayer as the capture substrate. Upon target recognition, the nanozymes catalyze the oxidation of 3,3',5,5'-tetramethylbenzidine (TMB), generating diffusible and positively charged TMB+ that spontaneously migrate into the hotpots of the negatively charged monolayer, enabling intense SERS enhancement free from spatial constraints. For p-Tau181 detection, the platform achieved remarkable detection limits of 50 fg/mL in standard solution and 100 fg/mL in artificial serum using a portable Raman spectrometer, providing a robust and versatile strategy for ultrasensitive detection of diverse disease-related proteins.