Design of δ-L-Lysinyl-L-glutamic Acid Dimer-Functionalized Peptides with Antifouling and Antienzymatic Degradation Capabilities for Robust Electrochemical Sensing of Proteins in Undiluted Clinical Sera

Nonspecific adsorption remains a persistent obstacle in biosensing applications, as it adversely impacts key performance indicators of biosensors such as sensitivity, specificity, and operational stability. Although conventional antifouling peptides have achieved much success, their practical utility is often constrained by susceptibility to enzymatic hydrolysis as natural enzymes exist in biological fluids. Herein, a robust nonfouling biosensor is proposed based on a newly designed peptide (δ-P) with both antifouling, antienzymatic degradation and recognition capabilities, functionalized with a δ-L-lysinyl-L-glutamic acid dimer. Crucially, the antifouling domain of the peptide is composed of a δ-L-lysinyl-L-glutamic acid dimer (glutamic acid with the lysine side chain), and the recognition domain specific for the protein ANXA1 is composed of D-type amino acids, and both of them are capable of resisting enzymatic degradation. Two types of proteases (alkaline protease and trypsin), which represent less site-specific and site-specific cleavage proteases, respectively, were tested to illustrate proteolysis. Taking advantages of the designed peptide, a highly sensitive and low-fouling electrochemical biosensor (with a linear range of 0.001-1000 ng mL^-1 and a detection limit of 0.32 pg mL^-1), capable of assaying protein ANXA1 in human sera, was prepared by attaching the peptide onto the electrode modified with a poly(3,4-ethylenedioxythiophene)-sodium alginate (PEDOT@SA) film and gold nanoparticles. More interestingly, the biosensor showed satisfactory accuracy for the detection of ANXA1 in undiluted clinical serum samples (verified by the ELISA method). It is anticipated that the peptide designing strategy presented in this work can be easily extended to the development of various robust antifouling biosensors capable of assaying targets in complex biological environments.