Electrochemical paper-based antigen sensing platform using plant-derived monoclonal antibody for detecting SARS-CoV-2

Abstract

The current approaches of diagnostic platforms for detecting SARS-CoV-2 infections mostly relied on adapting the existing technology. In this work, a simple and low-cost electrochemical sensing platform for detecting SAR-CoV-2 antigen was established. The proposed sensor combined the innovative disposable paper-based immunosensor and cost-effective plant-based anti-SARS-CoV-2 monoclonal antibody CR3022, expressed in Nicotiana benthamiana. The cellulose nanocrystal was modified on screen-printed graphene electrode to provide the abundant COOH functional groups on electrode surface, leading to the high ability for antibody immobilization. The quantification of the presence receptor binding domain (RBD) spike protein of SARS-CoV-2 was performed using differential pulse voltammetry by monitoring the changing current of [Fe(CN)₆]^3-/4- redox solution. The current change of [Fe(CN)₆]^3-/4- before and after the presence of target RBD could be clearly distinguished, providing a linear relationship with RBD concentration in the range from 0.1 pg/mL to 500 ng/mL with the minimum limit of detection of 2.0 fg/mL. The proposed platform was successfully applied to detect RBD in nasopharyngeal swab samples with satisfactory results. Furthermore, the paper-based immunosensor was extended to quantify the RBD level in spiked saliva samples, demonstrating the broadly applicability of this system. This electrochemical paper-based immunosensor has the potential to be employed as a point-of-care testing for COVID-19 diagnosis.

Keywords: Differential pulse voltammetry; Paper-based electrochemical immunosensor; Plant-based antibody; Point-of-care; SARS-CoV-2.

Graphical abstract

Fig. 1

The design in top view (A), side view (B), the operation (C) and the detection procedure (D) of paper-based electrochemical immunosensor.

Scheme 1

The process of mAb CR3022 covalent immobilization onto the electrode surface for electrochemical detection of SARS-CoV-2 RBD.

Fig. 2

Characterization of the modified electrodes in a step-by-step by EIS (A), CV (B), and the current response of 5 mM [Fe(CN)₆]^3−/4− in the absence and presence of 500 ng/mL of RBD that were performed by DPV (C).

Fig. 3

(A) DPV response in 5 mM [Fe(CN)₆]^3-/4- of the proposed immunosensor in different concentration of RBD in the range of 0.1 pg/mL to 500 ng/mL. (B) Relationship between Δ current and the concentration of RBD (inset is the calibration curve for proposed immunosensor).

Fig. 4

Cross-reactivity of the proposed immunosensor.