Label-free and reagent-less electrochemical detection of nucleocapsid protein of SARS-CoV-2: an ultrasensitive and disposable biosensor

Abstract

A novel selective and ultrasensitive sandwich biosensor was developed for the detection of nucleocapsid biomarkers of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). The biosensor fabrication strategy was composed of a combination of gold nanoparticles (AuNPs) electrodeposition and an aldehyde substituted thiophene monomer (3-Thi-Ald) electropolymerization. In this study, an effective surface design was achieved during biosensor fabrication by covalent immobilization of biorecognition elements. The Pthi-Ald polymer not only formed a surface immobilization matrix, but also supported the electrode surface conductivity with its conductive property. The immobilized antibodies on Pthi-Ald polymer could selectively capture nucleocapsid antigen via the specific immunoreaction that resulted in a blockage of the electron transfer through the working electrode because of the resistance of nucleocapsid protein. Electrochemical impedance spectroscopy (EIS), cyclic voltammetry (CV), and scanning electron microscopy (SEM) techniques were utilized to confirm the step-by-step fabrication procedure of the immunosensor. The developed immunosensor showed good selectivity, high storage stability, acceptable repeatability, and a good reproducibility for nucleocapsid detection. The AuNPs-Pthi-Ald polymer modified biosensor illustrated a good linear dynamic detection range between 0.0015 pg mL⁻¹ and 150 pg mL⁻¹ nucleocapsid concentration with a low detection limit of 0.48 fg mL⁻¹. Moreover, the immunosensor had excellent selectivity, stability, and performance in the analysis of artificial nasal secretion samples. This strategy provided a new aspect for the early screening of SARS-CoV-2 infection.