On-column enzymatic synthesis of melanin nanoparticles using cryogenic poly(AAM-co-AGE) monolith and its free radical scavenging and electro-catalytic properties

Abstract

In the present study we have demonstrated a green-route for the synthesis of melanin nanoparticles (Mel-NPs). To achieve this, a monolith column of poly(acrylamide-co-allylglycidyl ether) was synthesized and further modified to immobilize the enzyme tyrosinase, which was extracted from the corm of plant tuber Amorphophallus campanulatus. The immobilization of the enzyme was carried out in two steps. First, the epoxy groups present on the monolith surface were coupled with ethylene diamine followed by glutaraldehyde to introduce aldehyde moieties. In the second step, aldehyde functionalized monolith column was treated with enzyme solution for covalent bonding through Schiff's base formation. The physico-chemical characterization of monolith, exhibited ideal column characteristics like interconnected pore architecture, high flow rate, hydrophilicity and amiable mechanical strength. The convective flow of L-3,4-dihydroxyphenylalanine through the column brought about its conversion to Mel-NPs by biocatalytic activity of the immobilized enzyme. The spectral changes of the solution recorded in UV-visible region at regular time intervals showed that the synthesis of Mel-NPs occurred via formation of different intermediates. The size of the spherical Mel-NPs was in the range of 20 to 30 nm and its colloidal stability at different pH values was confirmed by measuring the zeta potential after a period of 8 weeks. FT-IR and TG analysis indicated that the bio-synthesized Mel-NPs showed characteristics similar to that of natural melanin. The IC₅₀ value for free-radical scavenging activity of Mel-NPs was calculated to be 24 μg mL⁻¹ using DPPH assay. Ferric-reducing action of Mel-NPs showed a concentration dependent increase under experimental conditions. Further, electro-catalytic activity of the glassy carbon electrode (GCE) modified with Mel-NPs was evaluated and was found to show a 2.4 fold enhancement in electro-chemical signal when compared with bare electrode. In conclusion, Mel-NPs synthesized by this novel approach can find applications in the development of antioxidant formulations, biosensor and other areas of interest.