Viral antigens detection has been gaining attention for early coronavirus detection to improve screening diagnostic reliability. Among them, nucleocapsid protein (NP) is an ideal biomarker related to early diagnostic detection once it is one of the proteins predominantly expressed with the virus infection and it can also be detected up to 1 day before clinical symptoms. In this way, we report an electrochemical immunoassay to detect nucleocapsid protein using screen-printed electrodes modified with carbon black/ chitosan suspension and capture antibodies (Ab1) targeting SARs-CoV-2. The NP was detected using chronoamperometry in a sandwich assay combining Ab1 and HRP-antibody (Ab2) for capturing and detection steps, respectively. For electrochemical detection, the 3,3′,5,5′-tetramethylbenzidine (TMB) was used as the redox probe. The Ab2 containing enzyme bound to NP and catalyzed the TMB oxidation, this electroactive compound was detected and the signal increased with the NP concentration. Amine groups of chitosan (CH) were activated by the glutaraldehyde to provide the covalent bond of the capture antibodies. For this purpose, 0.01, 0.05, 0.1, 0.25 and 0.5% (w/v) chitosan concentrations in 2.0 % of acetic acid were evaluated to establish the optimal polymer concentration to bind Ab1 and detect NP. The optimal percentage of CH was 0.05%. Another step of the sandwich assay was optimized; the concentration of glutaraldehyde (cross-linking) in three different concentrations, 1.0, 2.5, and 5.0 % (v/v). The last concentration showed a better ratio between the blank and 100 ng mL<sup>-1</sup> of NP, consequently ensuring a higher bound of antibodies and improving the analytical signal of current. The HRP-antibody concentration and incubation time for both NP and Ab1 have been studied to ensure the best performance of the assay, allowing the detection of different virus concentrations and early COVID-19 diagnosis. The developed device is a promising tool for point-of-care (POC) analysis.

Disposable screen-printed electrode modified carbon black and chitosan suspension aiming at the development of an electro chemical immunoassay for nucleocapsid protein

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