A heparin-modified palladium nanozyme for photometric determination of protamine

Heparin was employed as the stabilizing agent in the synthesis of peroxidase-mimicking Pd nanoparticles. The heparin-capped Pd nanozyme can act as both the signal amplifier and the selective binder of protamine. The most efficient nanozyme with the mean size of 3.5 nm consists of 70.8% metallic Pd0 and 29.2% Pd2+ species. Enzyme kinetic studies show that the Km values are 0.036 mM for 3,3′,5,5′-tetramethylbenzidine and 78 mM for H2O2. Protamine shows strong affinity to the heparin-capped Pd nanozyme, and induces an apparent aggregation of the nanoparticles. This results in a significant inhibition of the peroxidase-mimicking activities. Hence, the oxidation of TMB by H2O2 to a blue product with a maximum absorption at 652 nm is suppressed. Based on this finding, a photometric assay is developed for the determination of protamine. The linear response is in the concentration range 0.02 ~ 0.8 μg mL−1, and the limit of detection is 0.014 μg mL−1. This assay presents high selectivity toward other biological substances.