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

This work suggested that Cu(2+) 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 K(H)) to chelate multiple Cu(2+) ions. The resulting biotin-poly-(K(H)-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-(K(H)-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.