Highly sensitive and ultra-rapid antigen-based detection of SARS-CoV-2 using nanomechanical sensor platform

Abstract

The rapid spread of COVID-19 including recent emergence of new variants with its extreme range of pathologies create an urgent need to develop a versatile sensor for a rapid, precise, and highly sensitive detection of SARS-CoV-2. Herein, we report a microcantilever-based optical detection of SARS-CoV-2 antigenic proteins in just few minutes with high specificity by employing fluidic-atomic force microscopy (f-AFM) mediated nanomechanical deflection method. The corresponding antibodies against the target antigens were first grafted on the gold-coated microcantilever surface pre-functionalized with EDC-NHS chemistry for a suitable antibody-antigen interaction. Rapid detection of SARS-CoV-2 nucleocapsid (N) and spike (S1) receptor binding domain (RBD) proteins was first demonstrated at a clinically relevant concentration down to 1 ng/mL (33 pM) by real-time monitoring of nanomechanical signal induced by antibody-antigen interaction. More importantly, we further show high specific detection of antigens with nasopharyngeal swab specimens from patients pre-determined with qRT-PCR. The results take less than 5 min (swab to signal ≤5 min) and exhibit high selectivity and analytical sensitivity (LoD: 100 copies/ ml; 0.71 ng/ml of N protein). These findings demonstrate potential for nanomechanical signal transduction towards rapid antigen detection for early screening of SARS-CoV-2 and its related mutants.

Keywords: Antigens; Detection; Microcantilever; SARS-CoV-2; Sensitivity; Sensor.

Fig. 1

Optical detection scheme for SARS-CoV-2 S1 (RBD-Receptor Binding Domain) protein on a microcantilever surface.

Fig. 2

Schematic of cantilever biofunctionalization using EDC-NHS chemistry. Monoclonal antibodies for spike and nucleocapsid proteins were immobilized on functionalized microcantilever surface.

Fig. 3

Concentration dependant response curves for SARS-CoV-2 S1 and N proteins; cross-validation and control experiments were also performed by interacting both S1 and N proteins in different combinations on the cantilever surface to ascertain the specificity in the detection, (a) Deflection plots for S1 protein detection, (b) Deflection plots for N protein, (c) Table for deflection values for different concentrations of S1 and N protein. Anti-S/N protein: detection of N protein onto anti-S1 antibody conjugated cantilever (cross-validation); Anti-S/N–S mix: interaction of N and S1 protein mixture solution onto an anti-S1 antibody coated cantilever surface (cross-validation); anti-N/S protein: detection of S1 protein onto anti-N antibody conjugated cantilever (cross-validation); anti-N/S–N mix: interaction of S1 and N protein mixture solution onto an anti-N antibody conjugated cantilever surface. The measurements were performed on multiple cantilevers for each concentration. Though, the data represented here is for individual cantilever to demonstrate our sensor characteristics.

Fig. 4

Optical deflection measurement on patient samples with different Ct values. The curve represents deflection measurement for each Ct value along with the corresponding viral load (copies/ml) in the patient samples collected. The positive sample with the highest Ct value (39.7) yielded the lowest deflection (6.25 ± 3–4 nm) whereas maximum deflection (47.97 ± 3–4 nm) was exhibited for the patient sample having the lowest Ct value (13.18). The sensor did not show any measurable deflection for non-specific samples from MERS-CoV and Influenza A virus strain demonstrating specificity of the sensor, (b) Deflection curve for different Ct values with relative standard deviation (RSD) values. Error bars represent the standard error of three cantilevers, (c) Representation of deflection curve after the linear fitting, (d) Sensor stability measurement for a different period of time. The sensor exhibited stability in the data without losing any significant sensitivity for a period of fifteen days. The deflection and related RSD values have been provided in a tabulated form in Supplementary Information (Table S4).

Fig. 5

Curve for serial dilution measurement of patient sample for the determination of LoD. A series of eight different dilutions were used from a SARS-CoV-2 positive specimen with a cycle threshold (Ct) value of 13.18 (6 × 10⁹ copies/ml). The experiments were conducted on three individual microcantilevers. The error bar in the curve represents the standard error of the mean.