Selection, alkaline phosphatase fusion, and application of single-chain variable fragment (scFv) specific to NT-proBNP as electrochemical immunosensor for heart failure

Abstract

Heart failure has a high global prevalence, with symptoms such as breathlessness, fatigue, and swelling. Early detection is crucial, as the condition worsens over time and can be fatal. This study identified the single-chain variable fragment (scFv) that specifically binds to the heart failure biomarker N-terminal pro B-type natriuretic peptide (NT-proBNP) using biopanning techniques for the development of an alternative diagnostic tool. Ten clones were identified that bound to the target peptide, with two clones (scFv-16 and scFv-36) selected for further analysis. Soluble scFv-16 and scFv-36 were produced and fused with alkaline phosphatase (AP) for potential applications. The binding efficiency and specificity levels of scFv to natriuretic peptides were evaluated using surface plasmon resonance (SPR) analysis. The values of the dissociation constant (KD) for NT-proBNP of scFv-16, scFv-36, scFv-16-AP, and scFv-36-AP were in the range 3.72 × 10−7–3.42 × 10−8 M with high specificity. All constructed scFvs had specificity to NT-proBNP, while not binding to A-type (ANP) and C-type (CNP) natriuretic peptides. When AP was combined, the scFv had a slightly higher yield of expression. The enzyme activity of scFv-36-AP was observed first by the absorption at 405 nm at a minimum of 44 nM and then by the naked eye at a minimum of 88 nM. Additionally, the potential application of NT-proBNP binding scFv was preliminarily investigated using an electrochemical technique to directly detect NT-proBNP in phosphate buffer saline. The results revealed the limit of detection at 69.09 pg/mL, which was less than the cutoff value (150 pg/mL) to discharge patients or healthy people. These findings provided promising biomolecules for the development of a reliable and sensitive diagnostic tool for heart failure.