Structure-Guided Affinity Maturation of an Antibody Fragment for Gold-Surface Biosensing of N-Terminal Pro-B-Type Natriuretic Peptide

Rapid and specific detection of N-terminal pro-B-type natriuretic peptide (NT-proBNP) is critical for improving heart-failure diagnosis. In this study, a single-chain variable fragment (scFv) recognizing NT-proBNP and incorporating a gold-assembling linker (C12AP) was engineered and affinity-matured to enhance binding performance for gold-surface biosensing applications. Structure-guided docking of the C12AP–NT-proBNP complex informed semirational mutagenesis targeting residues D110 in VH-CDR3 and Y174 in VL-CDR1. Twenty-two scFv variants were expressed in Escherichia coli and functionally screened by ELISA using NT-proBNP as the analyte, human serum albumin as a specificity control, and the parental C12AP as a normalization reference. Eight variants were purified for quantitative biophysical evaluation. Microscale thermophoresis identified variant D110–57 as exhibiting significantly improved affinity relative to C12AP (p = 0.0174). Consistent with this result, molecular docking predicted a more favorable binding free energy for D110–57 (−254 kcal·mol–1) compared with C12AP (−209.6 kcal·mol–1), accompanied by additional stabilizing interactions involving VH-CDR2 and VL-CDR3. Molecular dynamics simulations confirmed the structural stability of the protein–ligand complexes, with both C12AP and D110–57 systems reaching equilibrium after ∼40 ns. Dynamic analyses revealed that key residues, particularly Asp110, undergo adaptive hydrogen bonding and salt bridge rearrangements, contributing to stable ligand coordination. To assess performance under biosensor-relevant conditions, surface plasmon resonance imaging was conducted using directed immobilization mediated by a histidine/serine-rich gold-assembling linker and a C-terminal His-tag. D110–57 exhibited concentration-dependent binding to NT-proBNP after immobilization on the gold surface. Kinetic analysis yielded a comparable KD value to that of the parental C12AP. Moreover, the variant maintained specificity, showing negligible cross-reactivity toward other natriuretic peptides. These results demonstrated that D110–57 combines enhanced affinity, preserved specificity, and compatibility with gold-surface immobilization, supporting its application as a recognition element for NT-proBNP biosensors.