Design, characterization, and validation of a high-affinity synthetic peptide as bioreceptor for label-free detection of prostate specific antigen.

Prostate-specific antigen (PSA), a serine protease produced by the prostate epithelial cells, is a key biomarker in prostate-associated diseases. While current diagnostic methods to detect prostate abnormalities such as PSA tests, digital rectal exams, prostate biopsies, etc., are widely used, they are often invasive, have poor selectivity and are time-consuming. Thus, an improved and simplified PSA detection strategy is required. In the current study, we report a novel peptide P1 specifically designed to target PSA. Intrinsic tryptophan fluorescence spectroscopy (TFS) was explored to validate the binding affinity between P1 and PSA. This approach, although effortless, has not been widely explored for binding studies. The presence of 7 tryptophan residues in PSA, with 2 of them in its binding site, led us to explore TFS for recognition of PSA-P1 interactions. Four novel synthetic peptide analogues were designed from a PSA-specific monoclonal antibody, with P1 (TFTTYYILDYA) exhibiting the strongest binding affinity towards PSA (-11.8 kcal/mol). Tryptophan Fluorescence spectroscopy (TFS) subsequently validated the strong binding affinity of P1 towards PSA with an apparent dissociation constant (KD) of 52 nM when PSA was titrated with different concentrations of P1, and a KD of 0.23 µM when P1 was titrated with varying concentrations of PSA. Secondary structure analysis revealed that P1 adopts a β-sheet conformation and exhibits notable thermal stability, withstanding temperatures as high as 205 °C, which renders it a stable bioreceptor for robust detection of PSA. The results suggest that P1 holds significant potential as a PSA-specific bioreceptor in biosensors. This is the first study of its kind. It ascertains that P1 is a viable, cost-effective, highly stable, robust, and selective peptide with strong potential to be a label-free bioreceptor for recognising PSA in various biological matrices. Its ability to form stable, selective and biocompatible interactions with PSA makes P1 a critical component in developing non-invasive and efficient detection systems.