A Robust Nonfouling Biosensor Based on an Engineered Multifunctional Monocyclic Peptide for Electrochemical Detection of Biomarkers in Human Blood

Electrochemical biosensors are susceptible to interference from biofouling of nonspecific proteins and the proteolytic hydrolysis of immobilized biomolecules by natural enzymes in complex biological media like blood. Therefore, enhancing the antifouling capability and the antienzymatic degradation ability is crucial for biosensors to perform in practical clinical samples. Herein, an electrochemical biosensing platform based on a multifunctional monocyclic peptide (MMCP) was developed, and the MMCP was designed with antifouling and antienzymatic degradation properties and the specific target (human epidermal growth factor receptor 2, HER2) recognition capability. The MMCP contains an antifouling part formed through covalent cyclization of a linear antifouling sequence (ECHHHHKHHHHCE) and a recognition d-peptide sequence (refffly) that can specifically recognize and bind HER2. Due to the cyclic structure and the adoption of d-amino acids, the MMCP can effectively resist the enzymatic degradation and shows significantly enhanced stability. The MMCP-based biosensor demonstrated outstanding antifouling performance and hydrolysis resistance properties in complex biological environments, and it was capable of sensitively detecting HER2 with a linear range of 1.0 pg mL^-1 to 1.0 μg mL^-1 and a low limit of detection of 0.35 pg mL^-1. Additionally, the assay results of the biosensor for HER2 in clinical blood samples were consistent with those obtained from the enzyme-linked immunosorbent assay (ELISA). The strategy of designing a specific peptide reported herein offers an effective way to construct robust biosensors for biomarker detection in complicated clinical samples.