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.