Characterization of horseradish peroxidase immobilized on PEGylated polyurethane nanoparticles and its application for dopamine detection

Abstract

In this work, we present a study of peroxidase adsorption on PEGylated polyurethane (PU-PEG) nanoparticles using a purified horseradish peroxidase (HRP). The influence of the immobilization on the catalytic activity of the enzyme was also investigated and the system was applied as a modified carbon paste electrode to dopamine determination in pharmaceutical products. HRP adsorption onto PU-PEG nanoparticles followed the Langmuir isotherm model reaching an adsorption plateau at the concentration of 22.5μg HRPmg−1 nanoparticles. An estimation theoretical calculation was done regarding the number of proteins molecules immobilized per nanoparticle, based on the molecular weight of the protein and the nanoparticles size. It was found that about 4400 HRP molecules were adsorbed to one PU-PEG nanoparticle, which corresponds to about 45% of the surface area of the nanoparticle. Transmission electron microscopy (TEM) performed on immuno-gold labeled samples showed the HRP adsorbed onto the surface of PEGylated polyurethane nanoparticles. HRP-adsorbed PU-PEG nanoparticles dispersions retained 50% of the enzyme activity even after 50 days of storage. Square-wave voltammetry (SWV) experiments were carried out to investigate the performance of the modified carbon paste electrode containing HRP-adsorbed PU-PEG nanoparticles and the analytical curve was linear for dopamine concentrations from 1.7×10−5 up to 1.9×10−3molL−1 (r=0.9997), with a detection limit of 2.0×10−6molL−1. The recovery of dopamine from pharmaceutical samples ranged from 93% to 107% and the results obtained using the modified carbon paste electrode and those obtained by the official method are in agreement at the 95% confidence level.