SARS-CoV-2 N protein IgG antibody detection employing nanoporous anodized alumina: A rapid and selective alternative for identifying naturally infected individuals in populations vaccinated with spike protein (S)-based vaccines

[EN] Methods to detect naturally infected individuals, especially during or after a pandemic, are valuable in populations with a high rate of vaccination. Having in mind that after the COVID-19 pandemic people has been vaccinated against the virus by Spike protein (S)-based vaccines, we present in this paper a novel nanosensor based on gated nanoporous anodic alumina (NAA) material to detect naturally infected individuals in populations with high rates of vaccination. The nanosensor developed is based on a protein-capped nanomaterial for the identification of IgG antibodies that can detect nucleocapsid protein (N) of SARS-CoV-2. The NAA material has been loaded with an indicator (Rhodamine B (RhB)) and the pores of the material have been blocked with SARS-CoV-2 nucleocapsid protein (N) attached to a specific aptamer. In presence of antibodies against this antigen, the pores are uncapped, triggering the dye release and a fluorescent signal as a result. The biosensor has been tested in vitro and simulated serum for IgG detection, proving a detection limit of 1 mu g/mL. Moreover, specificity assays with N proteins from other coronaviruses have proved the robustness and efficacy of this nanosensor. Finally, the system has been tested on samples from patients that contained SARS-CoV-2 antibodies, demonstrating its potential for the discrimination of individuals that have been vaccinated or infected by SARS-CoV-2 virus. This research was supported by projects PID2021-126304OB-C41, PID2021-128141OB-C22, and PID2021-122875OB-100 funded by MCIN/AEI/10.13039/501100011033/and by European Regional Development Fund-A way of doing Europe. This work was also funded by the Generalitat Valenciana (project no.2 RD 180/2020, CIPROM/2021/007) , Santander and CRUE Supera COVID-19 Fund (DIACOVID project) , the Universitat Politecnica de Valencia-Instituto de Investigacion Sanitaria La Fe (IIS-LaFe) (SARS-COV-2-SEEKER and VISION-COV projects) . R.M thanks to the European Union's Horizon EUROPE Research and Innovation Program (grant agreement No 101093042) . Y.E.-S. acknowledges the Ministerio de Universidades for his predoctoral grant (FPU20/05297) . E.C. thanks the Instituto de Salud Carlos III (Miguel Servet 2023 CP23/00086) . Scheme 1 done with BioRender.com . The use of clinical samples from patients was approved by the Drug Research Ethics Committee, CEIm of Hospital Universitari i Politecnic La Fe of Valencia (2021-012-1) . In all cases was obtained an informed oral consen