Development\nof a Dual-Epitope Nanobody-Based Immunosensor\nwith MXenes@CNTs@AuNPs for Ultrasensitive Detection of Rotavirus

Immunoassays have become essential tools for detecting\ninfectious\nviruses. However, traditional monoclonal antibody-dependent immunoassays\nare costly, fragile, and unstable, especially in complex media. To\novercome these challenges, we have developed cost-effective, robust,\nand high-affinity nanobodies as alternatives to monoclonal antibodies\nfor rapid detection applications. We engineered dual-epitope nanobody\n(NB) pairs and incorporated them into a sandwich immunosensor design\nto detect transmitted rotaviruses in rectal swabs and wastewater samples.\nTo further enhance sensitivity, we synthesized an advanced two-dimensional\nmaterial, MXenes@CNTs@AuNPs, which offers an extensive specific surface\narea that supports the enrichment and immobilization of NBs. This\nintegration with catalase-modified magnetic probes facilitates signal\ngeneration. Subsequently, our sensor achieved a detection limit of\n0.0207 pg/mL for the rotavirus VP6 antigen, significantly outperforming\ncommercial antigen kits with a sensitivity enhancement of 3.77 ×\n10<sup>5</sup>-fold. The exceptional sensor performance extended to\nspecificity, repeatability, stability, and accuracy across various\nsample types, establishing it as a promising tool for rotavirus detection.\nThis research outlines a viable strategy for creating a robust and\nultrasensitive analytical nanoprobe, thereby addressing the critical\nneed for efficient and reliable viral detection methods in various\nenvironments.