Nanostructured electrochemical biosensor modified with a plant-derived protein for serine protease detection in...

This study introduces, for the first time, a bioelectrochemical device utilizing a trypsin inhibitor protein (EcTI) from Enterolobium contortisiliquum seeds, designed to interact with serine proteases, including prostate-specific antigen (PSA), which is involved in the progression of prostate cancer. The label-free biosensor was engineered via covalent immobilization of EcTI onto a nanostructured conductive film composed of polypyrrole (PPy) and carboxylated multi-walled carbon nanotubes (MWCNTs), integrated with amino-functionalized titanium dioxide (TiO₂) nanoparticles. The EcTI bioactivity and biosensor performance were investigated using PSA standards, human serum samples, and PC-3 metastatic prostate cancer cells, employing cyclic voltammetry (CV) and electrochemical impedance spectroscopy (EIS). In parallel, film morphology and chemical functionalization were characterized by atomic force microscopy (AFM) and attenuated total reflectance Fourier transform infrared (ATR-FTIR) spectroscopy, respectively. Serine-class proteolytic enzymes exhibited differential recognition signatures in serum from prostate cancer patients and PC-3 cell assays, enabling a comparative analysis relative to isolated PSA measurements. In serological tests, the biosensor showed linearity from 0.50 to 23.28 ng/mL, with a limit of detection (LOD) of 0.56 ng/mL and a limit of quantification (LOQ) of 1.71 ng/mL. In PC-3 cell assays, the response was linear from 4.60 × 10¹ to 4.60 × 10⁶ cells/mL, with a LOD of 0.46 cells/mL and LOQ of 1.40 cells/mL. The innovative use of EcTI enabled the recognition of serine proteases, such as PSA, with high reproducibility and signal repeatability, underscoring the system's potential for precise prostate cancer diagnosis.