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

Lin Liao¹Zhaoxiong Li¹Zhanhong Liu¹Bin Qiu²Wangyuan Guo^1*

• ¹The First People's Hospital of Chenzhou, Chenzhou, China
• ²Fuzhou University, Fuzhou, China

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 H₂O₂, 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 (0.1–1500 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.