Silver Nanotripod Array-Based Sandwich Immunosensor for Sensitive SERS Detection of SARS-CoV-2 Spike Protein and Multiplexed Protein Samples

The COVID-19 pandemic caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) has significantly impacted public health and social stability, highlighting the urgent need for rapid diagnostic methods to ensure timely clinical intervention. Herein, we develop a sensitive surface-enhanced Raman scattering (SERS) immunosensor based on a silver nanotripod (AgNT) array sandwich structure for simulating early SARS-CoV-2 detection. AgNT arrays modified with SARS-CoV-2 antibody serve as a capture substrate; antibody-modified gold nanoparticles (AuNPs) functionalized with dye molecules act as detection probes. Upon binding to analyte, these components form an AuNPs@AgNT sandwich immunosensor generating enhanced SERS signals for detection. The detection limit of AuNPs@AgNT for SARS-CoV-2 spike protein in phosphate-buffered saline (PBS) samples and saliva samples could reach 0.1 and 0.77 pg mL −1 , respectively. This sandwich immunosensor demonstrates excellent reproducibility with a relative standard deviation lower than 8% and maintains satisfactory detection specificity even in various saliva samples and other proteins. Furthermore, by integrating machine learning technology, the sandwich immunosensor enables accurate discrimination of multi-target samples. These results illustrate the promise of the SERS sandwich immunosensor for the early SARS-CoV-2 spike protein detection, which has a remarkable significance for the virus detection in the future. Graphical The front cover shows a surface-enhanced Raman scattering (SERS) immunosensor based on Ag nanotripod (AgNT) arrays. Au nanoparticles (AuNPs) are loaded with reporters (4-MBA, DTNB, TFMBA) and antibodies (Anti). The AgNT substrate is modified with 3-MPA and Anti. The immunosensor combined with machine learning enables detection of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) spike protein and distinction of tumor necrosis factor-α (TNF-α) and interleukin-1 (IL-1) in mixed samples.