Development of a Single-Chain Variable Fragment of CR3022 for a Plasmonic-Based Biosensor Targeting the SARS-CoV-2 Spike Protein

Abstract

Two years after SARS-CoV-2 caused the first case of COVID-19, we are now in the "new normal" period, where people's activity has bounced back, followed by the easing of travel policy restrictions. The lesson learned is that the wide availability of accurate and rapid testing procedures is crucial to overcome possible outbreaks in the future. Therefore, many laboratories worldwide have been racing to develop a new point-of-care diagnostic test. To aid continuous innovation, we developed a plasmonic-based biosensor designed explicitly for portable Surface Plasmon Resonance (SPR). In this study, we designed a single chain variable fragment (scFv) from the CR3022 antibody with a particular linker that inserted a cysteine residue at the second position. It caused the linker to have a strong affinity to the gold surface through thiol-coupling and possibly become a ready-to-use bioreceptor toward a portable SPR gold chip without purification steps. The theoretical affinity of this scFv on spike protein was -64.7 kcal/mol, computed using the Molecular Mechanics Generalized Born Surface Area (MM/GBSA) method from the 100 ns molecular dynamics trajectory. Furthermore, the scFv was produced in Escherichia coli BL21 (DE3) as a soluble protein. The binding activity toward Spike Receptor Binding Domain (RBD) SARS-CoV-2 was confirmed with a spot-test, and the experimental binding free energy of -10.82 kcal/mol was determined using portable SPR spectroscopy. We hope this study will be useful in designing specific and low-cost bioreceptors, particularly early in an outbreak when the information on antibody capture is still limited.

Keywords: SARS-CoV-2; molecular dynamics; plasmonic-based bioreceptor; portable SPR; scFv.

Figure 1

Molecular design of scFv.

Figure 2

Structure model of scFv in blue and RBD of SARS-CoV-2 in red (A); Model assessed by Ramachandran plot (B) and z-score analysis (C).

Figure 3

RMSD of protein backbone (A) Residual RMSF profile throughout 100 ns of simulation (B).

Figure 4

SASA analysis of cysteine residue in the linker.

Figure 5

Molecular interaction between scFv in blue and RBD in red (A) Hydrogen bond is depicted with a green dashed line; the salt bridge is depicted with an orange dashed line, and hydrophobic interactions are depicted with a pink dashed line (B).

Figure 6

(A). SDS-PAGE electropherogram (1) the scFvs (2) periplasmic protein of E. coli BL21 (DE3) without recombinant plasmid IPTG induced (3) and IPTG uninduced (B). Spot test Analysis (1) 0.5 mg/mL scFv (2) periplasmic protein of E. coli BL21 (DE3) without recombinant plasmid (3) 0.5 mg/mL BSA in the third lane (B).

Figure 7

(A) Preparation of the SPR sensor chip: immobilization of scFv, blocking with BSA, and response signal scFv-RBD interactions, (B) magnification image of SPR sensorgram scFv-RBD binding interactions, (C) binding interaction between scFv and various concentrations of RBD, (D) Non-linear fitting of experimental data by Hills and Dubinin-Radushkevich adsorption isotherm model, and (E) SPR dynamic response of IgY to 100 ng/mL SARS-CoV-2 RBD.

Figure 8

(A) SPR response dynamics and (B) bar chart of scFv response to AI, IB, and their mixture with Spike RBD of SARS-CoV-2 (SC2).

Figure 9

SPR response dynamics of the developed chip sensor to nasopharyngeal samples.