Enhanced susceptibility of SARS-CoV-2 spike RBD protein assay targeted by cellular receptors ACE2 and CD147: Multivariate data analysis of multisine impedimetric response.

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) enters the cells through the binding of spike protein to the host cell surface-expressing angiotensin-converting enzyme 2 (ACE2) or by endocytosis mediated by extracellular matrix metalloproteinase inducer (CD147). We present extended statistical studies of the multisine dynamic electrochemical impedance spectroscopy (DEIS) revealing interactions between Spike RBD and cellular receptors ACE2 and CD147, and a reference anti-RBD antibody (IgG2B) based on a functionalised boron-doped diamond (BDD) electrode. The DEIS was supported by a multivariate data analysis of a SARS-CoV-2 Spike RBD assay and cross-correlated with the atomic-level information revealed by molecular dynamics simulations. This approach allowed us to study and detect subtle changes in the electrical properties responsible for the susceptibility of cellular receptors to SARS-CoV-2, revealing their interactions. Changes in electrical homogeneity in the function of the RBD concentration led to the conclusion that the ACE2 receptor delivers the most homogeneous surface, delivered by the high electrostatic potential of the relevant docking regions. For higher RBD concentrations, the differences in electrical homogeneity between electrodes with different receptors vanish. Collectively, this study reveals interdependent virus entry pathways involving separately ACE2, CD147, and spike protein, as assessed using a biosensing platform for the rapid screening of cellular interactions (i.e. testing various mutations of SARS-CoV-2 or screening of therapeutic drugs).

Performance of electrochemical immunoassays for clinical diagnostics of SARS-CoV-2 based on selective nucleocapsid N protein detection: Boron-doped diamond, gold and glassy carbon evaluation.

The 21st century has already brought us a plethora of new threats related to viruses that emerge in humans after zoonotic transmission or drastically change their geographic distribution or prevalence. Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) was first spotted at the end of 2019 to rapidly spread in southwest Asia and later cause a global pandemic, which paralyzes the world since then. We have designed novel immunosensors targeting conserved protein sequences of the N protein of SARS-CoV-2 based on lab-produced and purified anti-SARS-CoV-2 nucleocapsid antibodies that are densely grafted onto various surfaces (diamond/gold/glassy carbon). Titration of antibodies shows very strong reactions up to 1:72 900 dilution. Next, we showed the mechanism of interactions of our immunoassay with nucleocapsid N protein revealing molecular recognition by impedimetric measurements supported by hybrid modeling results with both density functional theory and molecular dynamics methods. Biosensors allowed for a fast (in less than 10 min) detection of SARS-CoV-2 virus with a limit of detection from 0.227 ng/ml through 0.334 ng/ml to 0.362 ng/ml for glassy carbon, boron-doped diamond, and gold surfaces, respectively. For all tested surfaces, we obtained a wide linear range of concentrations from 4.4 ng/ml to 4.4 pg/ml. Furthermore, our sensor leads to a highly specific response to SARS-CoV-2 clinical samples versus other upper respiratory tract viruses such as influenza, respiratory syncytial virus, or Epstein-Barr virus. All clinical samples were tested simultaneously on biosensors and real-time polymerase chain reactions.