High sensitivity perovskite-tagged nanobody electrochemiluminescent immunosensor for spike (S1) protein biomarker-based persistent viral reservoir detection

Persistent viral reservoirs (PVR) of SARS-CoV-2 (or long COVID-19) and latent COVID-19 disease have been of great concern to clinicians. Several studies have identified spike protein (S1) as a definitive biomarker for the early detection of persistent viral reservoirs and latent COVID-19. A novel sandwich-format electrochemical immunosensor integrating a nanocomposite material was engineered for rapid and sensitive latent COVID-19 detection. The platform structure, AuSPE||strep|Nb₁|BSA|biotin|S1|strep-LiSmZrO₃-Nb₂ + BSA (AuSPE = gold screen-printed electrode, strep = streptavidin-thiol, Nb₁ = primary nanobody, Nb₂ = secondary nanobody, BSA = bovine serum albumin, and spike (S1) protein = S1), featured a disposable AuSPE modified with strep to anchor a biotinylated camelid Nb₁ specific to the spike protein. A Nb₂ conjugated to streptavidin-labelled LiSmZrO₃ perovskite completed the sandwich complex, enhancing both affinity and signal transduction. Electrochemical responses of the sensor were studied via electrochemiluminescent (ECL) signal transduction. The S1-sensitive sandwich immunosensor had a detection range of 0-1000 pg mL^-1 with a limit of detection of 0.04 pg mL^-1 via ECL. As feasibility studies, commercial spike protein in buffered solutions and human serum, highlighting the potential for the immunosensor to be used in SARS-CoV-2 patients and PVRs. The nanobody sandwich immunosensor showed excellent stability, selectivity, sensitivity, and reproducibility. The immunosensor serves as a broad PVR for a SARS-CoV-2 screening tool, detecting elevated S1 levels to enable early, targeted diagnostics.