Structural tuning of bridging linkers on M13 phages for high-affinity detection of enterovirus A71

Enterovirus A71 (EV-A71), the principal etiological agent of hand, foot, and mouth disease, poses a serious public health concern because infection can induce severe and potentially fatal neurological complications through direct invasion and inflammation of the central nervous system. Current detection methods lack rapid and sensitive analytical capabilities, particularly in the context of wastewater surveillance. This study reports a PEG4-biotin-functionalized M13 bacteriophage engineered as a molecular receptor for the colorimetric detection of EV-A71. VP1-specific phage ligands were identified via phage display and comprehensively characterized using matrix-assisted laser desorption/ionization time-of-flight (MALDI-TOF), attenuated total reflectance-Fourier transform infrared spectroscopy (ATR-FTIR), and transmission electron microscopy (TEM). The optimized biosensor demonstrated strong linearity for VP1 (0.03-3.91 nM, R² = 0.969) and intact virions (10²-10⁶ PFU/mL, R² = 0.974), achieving detection limits of 0.22 nM and ∼10² PFU/mL, respectively. These findings underscore the potential of the PEG4-biotinylated M13 platform for rapid, sensitive, and on-site enterovirus monitoring in public health surveillance systems.