Nanobody Coupled with Au Nanoparticle Cluster to Detect Survivin Protein by Lateral Flow Immunoassay.

The monoclonal antibody (mAb) used in a traditional Lateral Flow Immunoassay (LFIA) has many limitations, particularly related to its poor affinity, specificity, and stability. In this study, a nanobody (VHH) was developed with improved tolerability and higher specificity for the target. The strong binding affinity between VHH and survivin was emphasized by simulated docking, demonstrating the feasibility and effectiveness of replacing mAb in the detection of urinary survivin based on LFIA. In addition, the superior performance of VHH compared with mAb under extreme conditions was highlighted. To further enhance the detection performance, VHH was conjugated with an Au nanoparticle cluster (AuNP cluster). The pore size structure of the AuNP cluster led to an increased abundance of AuNPs, which amplified the signal while providing adequate adsorption sites for VHH coupling, thereby achieving a synergistic enhancement in the sensitivity and specificity of the AuNP cluster-VHH probe. Consequently, the detection limit for survivin was established at 3.90 ng/mL, facilitating noninvasive detection and monitoring in early-stage bladder cancer and during post-treatment surveillance. This insight underscores the key role of VHH in enhancing the performance of LFIA and the potential of developing a promising detection tool based on urinary biomarkers.