Isothermal signal amplification-mediated nanozyme capture on DNA tetrahedra for ultrasensitive amperometric immunoassay of Epstein–Barr virus latent membrane protein 1

The overexpression of latent membrane protein 1 (LMP-1), a key oncoprotein encoded by the Epstein-Barr virus (EBV), is closely associated with the development and progression of nasopharyngeal carcinoma (NPC), making it a valuable biomarker for early diagnosis and prognosis. Herein, we report a highly sensitive amperometric immunoassay method for LMP-1 detection based on the strand displacement amplification (SDA)-mediated capture of nanozyme on a DNA tetrahedron (TDN)-modified electrode. In detail, a sandwich immunoassay was carried out on a microplate, followed by sequential capture of streptavidin and biotin-labeled SP-HP, which then initiated SDA in the presence of a nicking enzyme and DNA polymerase. The resulting trigger DNA hybridized with TDN-anchored hairpin probes, exposing terminal digoxin moieties that captured anti-digoxin antibody modified AuPt alloy nanozymes. The nanozymes catalyzed the oxidation of TMB in the presence of H 2 O 2 , generating electroactive oxidized products that were subsequently reduced at the electrode to yield a measurable current signal. The integration of TDNs, SDA, and AuPt nanozymes significantly enhanced sensitivity, achieving a detection limit as low as 47 fg mL -1 and a wide linear range (0.1–1,500 pg mL -1 ). The developed method also demonstrated excellent specificity, reproducibility, and applicability in spiked biological samples. This work presents a promising strategy for ultrasensitive and specific detection of EBV-related proteins and expands the utility of nanozyme-based electrochemical immunoassays for clinical diagnostics.