Development of method for SARS-CoV-2 spike protein determination in saliva samples at fg mL−1 levels using electrochemically controlled preconcentration and molecularly imprinted polypyrrole sensor

Abstract

The present study describes a novel approach for designing a highly sensitive and selective electrochemical sensor for the detection of SARS-CoV-2 in saliva samples by sensing the S-protein, using an electrochemically controlled preconcentration. The sensor is based on electropolymerized molecularly imprinted polypyrrole (PPy) onto a screen-printed carbon electrode (MIP/PPy). The morphology of the MIP/PPy sensor was evaluated by SEM and AFM. The selectivity of the MIP/PPy compared to NIP/PPy was assessed by rebinding S-protein, followed by the monitoring of the redox probe ([Fe(CN)₆]^3−/4−). A substantial decrease in the anodic peak current of the redox probe was noticed when using the MIP/PPy. The detectability of the sensor was greatly improved (c.a 1,000,000 fold) by using controlled electrochemical preconcentration of S-protein at +0.6 V for 1.0 min in the rebinding process. Under this condition, a low limit of detection of 6.8fg mL⁻¹ and an analytical curve in the range of 50.0–125.0 fg mL⁻¹ (R² = 0.996) was obtained. The reproducibility for 10 authentic sensors was highly satisfactory with a relative standard deviation (%,RSD) of 2.8 %. Upon preparation, the sensor can be stored for up to 14 days under refrigeration, with a response efficiency greater than 85 %. The MIP/PPy exhibited high selectivity toward S-protein compared to antitrypsin, human serum albumin (HAS), transferrin, HIgG, and lysozyme. The sensor was applied for detecting SARS-CoV-2 virus through S-protein in infected patients’ saliva samples and validated by RT-PCR. In addition, the satisfactory recovery (91 to 103 %) in S-protein spiked negative samples attested to the free-interference analysis.