Double-antibody-based nano-biosensing system for the onsite monitoring of SARS-CoV-2 variants

The fast and reliable diagnosis of COVID-19 is the foremost priority for promoting public health interventions. Therefore, double-antibody-based immunobiosensor chips were designed, constructed, and exploited for clinical diagnosis. Gold nanoparticles/tungsten oxide/carbon nanotubes (AuNPs/WO₃/CNTs) were used as the active working sensor surface to support the chemical immobilization of a mixture of SARS-CoV-2 antibodies (anti-RBD-S and anti-RBD-S-anti-Llama monoclonal antibodies). The morphology and chemical functionalization of the fabricated disposable immunochips was characterized using scanning electron microscopy (SEM), Fourier transform infrared (FTIR) spectroscopy, cyclic voltammetry (CV), and electrochemical impedance spectroscopy (EIS). After full assay optimization, the immunobiosensor showed a high sensitivity to detect SARS-CoV-2-S protein with limits of detection and quantification of 1.8 and 5.6 pg/mL, respectively. On the other hand, for the SARS-CoV-2 whole virus particle analysis, the detection and quantification limits were determined to be 5.7 and 17 pg/mL, respectively. The biosensor showed a highly selective response toward SARS-CoV-2, even in the presence of influenza, nontargeting human coronaviruses, and Middle East respiratory syndrome coronavirus (MERS-CoV). The immunochips exhibited distinct responses toward the variants of concern: B.1>C.36.3>Omicron> Delta> Alpha coronavirus variants. For biosensor validation, twenty-nine clinical specimens were analyzed, and the impedimetric responses were positively detected for two Delta samples, eighteen Omicron samples, and six B.1-type samples in addition to three negative samples. Eventually, the immunobiosensor was fabricated in the form of ready-to-use chips capable of sensitive detection of virus variants, especially variants of concern (VOC) and interest, in a specimen within 15 min. The chips provided instantaneous detection with the direct application of clinical samples and are considered a point-of-care device that could be used in public places and hot spots.