Sensitive\nElectrochemical and Thermal Detection of\nHuman Noroviruses Using Molecularly Imprinted Polymer Nanoparticles\nGenerated against a Viral Target

Norovirus (NoV) is\nthe predominant cause of foodborne illness globally;\ncurrent detection methods are typically expensive, have inadequate\nsensitivities, and utilize biological receptors with poor stability.\nTherefore, accurate, cost-effective, and highly stable detection methods\nare needed to screen for NoV in foods. We developed molecularly imprinted\npolymer nanoparticles (nanoMIPs) to detect NoV using a small target\nepitope (12 amino acids) with a solid-phase synthesis approach. The\nperformance of three batches of nanoMIPs with varying monomer compositions\n(nanoMIP-1, -2, and -3) were compared both experimentally and computationally.\nSurface plasmon resonance examined nanoMIP binding affinity to norovirus\nvirus-like particles (NoV-LPs), whereby nanoMIP-1 had the lowest K<sub>D</sub> value of 0.512 μM. This is significant, as traditional\ntargets for generation of norovirus ligands previously reported were\ngenerated against drastically larger norovirus capsid segments that\nhave limitations in ease of production. Further, an electrochemical\nsensor was developed by covalently attaching the nanoMIPs to glassy\ncarbon electrodes. In agreement with our predictions from density\nfunctional theory simulations, electrochemical impedance spectroscopy\nshowed a sensitive response toward NoV-LPs for nanoMIP batches tested;\nhowever, nanoMIP-1 was optimal, with an excellent detection limit\nof 3.4 pg/mL (1.9 × 10<sup>5</sup> particles/mL). Due to its\nexceptional performance, nanoMIP-1 was immobilized to screen-printed\nelectrodes and utilized within a thermal sensor, where it exhibited\na low detection limit of 6.5 pg/mL (3.7 × 10<sup>5</sup> particles/mL).\nCrucially, we demonstrated that nanoMIP-1 could detect NoV in real\nfood samples (romaine lettuce) by using electrochemical and thermal\nsensors. Consequently, the study highlights the exceptional potential\nof nanoMIPs to replace traditional biological materials (e.g., antibodies)\nas sensitive, versatile, and highly stable receptors within NoV sensors.