Rational design and molecular docking-guided screening of high-affinity β-glucosidase affibodies for application in a...

Intestinal microbial β-glucosidase serves as a key functional biomarker associated with gut dysbiosis and colorectal cancer (CRC) progression. However, the development of rapid and sensitive point-of-care testing (POCT) methods for β-glucosidase has been limited by the lack of stable and high-affinity recognition elements. In this study, we combined AlphaFold3-based structural prediction with molecular docking to rationally engineer affibodies targeting β-glucosidase. Key binding residues, including Arg-35 and Phe-17, were precisely identified and subjected to saturation mutagenesis for directed optimization. Molecular dynamics simulations demonstrated that the optimized mutants exhibited enhanced conformational stability, with RMSD values ranging from 1.211 to 1.233 Å, outperforming the wild-type affibody. Among the engineered variants, Mut2 showed excellent thermal stability (Tm = 67.4 °C) and strong binding affinity at the micromolar level. Based on the optimized affibody, a sandwich-format lateral flow immunoassay (LFIA) platform was successfully developed for rapid β-glucosidase detection. The assay achieved a detection limit of 5 μg/mL (59 nM) within 15 min, together with good specificity and repeatability (CV = 3.57%). This work provides a promising strategy for the development of stable non-antibody recognition elements and establishes a rapid LFIA platform for potential early screening of intestinal malignancies.