Fluorescent Immunoassay with a Copper Polymer as the Signal Label for Catalytic Oxidation of O-Phenylenediamine

Abstract

This work suggested that Cu2+ ion coordinated by the peptide with a histidine (His or H) residue in the first position from the free N-terminal reveals oxidase-mimicking activity. A biotinylated polymer was prepared by modifying His residues on the side chain amino groups of lysine residues (denoted as KH) to chelate multiple Cu2+ ions. The resulting biotin-poly-(KH-Cu)20 polymer with multiple catalytic sites was employed as the signal label for immunoassay. Prostate specific antigen (PSA) was determined as the model target. The captured biotin-poly-(KH-Cu)20 polymer could catalyze the oxidation of o-phenylenediamine (OPD) to produce fluorescent 2,3-diaminophenazine (OPDox). The signal was proportional to PSA concentration from 0.01 to 2 ng/mL, and the detection limit was found to be eight pg/mL. The high sensitivity of the method enabled the assays of PSA in real serum samples. The work should be valuable for the design of novel biosensors for clinical diagnosis.

Progress in the Development of Biosensors Based on Peptide–Copper Coordination Interaction

“…The electrochemical and fluorescent methods show a detection limit of 0.40 and 1.00 pg/mL, respectively. Meanwhile, it was found that the Cu 2+ complexes performed with the His1-containing peptides reveal high activity for the catalytic oxidation of OPD to produce fluorescent 2,3-diaminophenazine (OPDox) and maintained excellent selectivity for PSA detection even in the presence of high concentration of interfering proteins [ 88 ]. Based on the difference in the redox potential and current of Cu 2+ before and after being coordinated by the His1- or His3-containing peptide, Feng et al proposed a general, label-free, and homogeneous electrochemical biosensor for the detection of protease activity [ 89 ].…”