Electrochemical Biosensor Utilizing Low-Susceptibility Macrocyclic Stapled Peptide to Mitigate Biofouling for Reliable Protein Detection in Human Serum

Antifouling peptide surfaces have garnered increasing attention due to their immense potential across various biochemical fields. Cyclic peptides, in particular, demonstrate greater resistance to nonspecific substance adsorption compared to regular linear peptides. Herein, inspired by the macrocyclization method, we introduced two non-natural amino acids (<i>R8</i> and <i>S5</i>) to cyclize the typical low-fouling sequence (EKEKEK) head-to-tail, thereby protecting terminal amino acids and reducing proteolytic susceptibility. This approach resulted in the formation of a novel stapled peptide (SP), which resisted protease hydrolysis and enhanced the antifouling capabilities of the SP-based biosensor compared to the conventional linear peptide (LP), demonstrated by electrochemical testing and fluorescence imaging experiments. Theoretically, molecular dynamics simulations were employed to calculate binding energies between the peptides and carboxypeptidase Y (CPY), and the results showcased a stronger binding affinity of LP with CPY, further confirming the lower proteolytic susceptibility of the engineered SP. Remarkably, the SP-based biosensor demonstrated high sensitivity in detecting the model target of carcinoembryonic antigen, with a limit of detection of 0.49 pg/mL. Moreover, clinical serum samples analyzed using the SP-based biosensor showed excellent concordance with hospital diagnostic methods, underscoring its exceptional accuracy and further highlighting the superiorities of the engineered SP structures.