Epitope-imprinted nanoparticles prepared by controlled solid-phase synthesis for low-nanomolar-affinity SARS-CoV-2 spike recognition and direct non-faradaic impedance sensing in oral fluid.

Herein we present molecularly imprinted nanoparticles (MIN) as synthetic antibodies with nanomolar affinity (4.57 ± 0.01 nM) targeting the spike protein of the SARS-CoV-2. The peptide corresponding to the residues 470-TEIYQAGST-478 was selected as the template to produce nanoparticles via solid-phase epitope imprinting, using a water-soluble photocleavable iniferter. MIN with different monomer compositions were screened by surface plasmon resonance (SPR) to identify the formulation with the highest affinity for the target protein, finding that the monomer N-(4-hydroxyphenyl) acrylamide (HPAm) made a significant contribution to achieving high affinity binding. Morphological and physicochemical analysis of the nanoparticles revealed irregular, spherical particles with diameters of 130.7 ± 9.4 nm with zeta potential values of 33.2 ± 57.4 mV. MIN were immobilised on interdigitated gold electrodes to fabricate sensors for spike protein detection based on non-faradaic impedance, simplifying the sensing protocol by eliminating the need for redox probes and reference electrodes, making it more suitable for point-of-care (PoC) applications. The sensor proved capable of directly determining the target protein in diluted samples without any further preparation, exhibiting a LOD and a LOQ of 15.3 pM and 35.7 pM, respectively, in oral fluid. Sensor selectivity was demonstrated through a competitive assay using the target peptide, its scrambled sequence (IYTATQGES), and the envelope protein as competitors, with only the target peptide showing significant competition. The methodology presented here constitutes a versatile platform with that can be readily extended to peptides derived from other viruses for the development of new miniaturised PoC devices.