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This study presents the synthesis of nanostructured surfaces via nanosphere lithography for the design of biosensors in order to detect pharmaceutical compounds. The result is a higher surface area available for the immobilization of the enzymes improving the analytical performances of the biosensors. The chosen model enzyme was Horseradish peroxidase, which was immobilized by coupling via the click chemistry reaction, an approach that maintains the tridimensional configurations and preserves its biological activity. This strategy could be applied for the immobilization of antibodies and antigens for the design of immunosensors involved in clinical diagnosis.