Affinity maturation of anti-NT-proBNP single-chain variable fragment (scFv) and biophysical characterization for heart failure biosensor application

Rapid, specific detection of N-terminal pro-B-type natriuretic peptide (NT-proBNP) enhances heart-failure diagnosis. An NT-proBNP–binding single-chain variable fragment (scFv) bearing a gold-assembling linker (C12AP) was engineered and affinity-matured to serve as a biosensor recognition element. Docking of C12AP–NT-proBNP complexes guided semi-rational mutagenesis at D110 (VH-CDR3) and Y174 (VL-CDR1). Twenty-two variants were expressed and screened by ELISA against NT-proBNP with human serum albumin as a specificity control and C12AP for normalization. Eight candidates were purified for quantitative analyses. Microscale thermophoresis (MST) identified variant D110-57 as significantly improved over C12AP ( p = 0.0174). Docking predicted a more favorable binding energy for D110-57 (−254 kcal·mol -1) versus C12AP (−209.6 kcal·mol -1), with additional contacts in VH-CDR2 and VL-CDR3. To emulate biosensor conditions, surface plasmon resonance imaging (SPRi) employed a self-assembling histidine/serine-rich linker and a C-terminal His tag to orient scFvs on gold. D110-57 exhibited concentration-dependent binding with concordance between steady-state and kinetic analyses (affinity K D = 5.167 nM; kinetic K D = 6.5 nM; k a = 5.396 × 10^6 M -1·s -1; k d = 0.0351 s -1). Specificity was retained, with minimal binding to A- and C-type natriuretic peptides. Overall, D110-57 shows enhanced affinity and specificity and is compatible with gold-surface immobilization, supporting application in NT-proBNP point-of-care biosensors.