RESEARCH ARTICLE Copyright © 2011 American Scientific Publishers All rights reserved Printed in the United States of America Journal of Nanoscience and Nanotechnology Vol. 11, 1–7, 2011 Increasing the Antibacterial Effect of Lysozyme by Immobilization on Multi-Walled Carbon Nanotubes Daniele Merli 1 , Monica Ugonino 1 , Antonella Profumo 1 , Maurizio Fagnoni 2 , Eliana Quartarone 3 , Piercarlo Mustarelli 35∗ , Livia Visai 45 , Marco S. Grandi 6 , Pietro Galinetto 6 , and Patrizia Canton 7 1 Department of General Chemistry, University of Pavia, Via Taramelli 12, 27100 Pavia, Italy 2 Department of Organic Chemistry, University of Pavia, Via Taramelli 10, 27100 Pavia, Italy 3 Department of Physical Chemistry, University of Pavia, Via Taramelli 16, 27100 Pavia, Italy 4 Biochemistry Department, University of Pavia Viale Taramelli 3/b 27100 Pavia 5 Center for Tissue Engineering (CIT), University of Pavia, Via Ferrata 1, 27100 Pavia, Italy 6 Department of Physics “A. Volta”, University of Pavia, Via Bassi 6, 27100 Pavia, Italy 7 Dipartimento di Chimica Fisica, Università Ca’ Foscari Venezia, Via Torino 155/b, 30170 Venezia-Mestre, Italy We report a facile strategy to obtain multiwalled carbon nanotubes (MWCNTs) functionalized with covalently bonded lysozyme. The functionalization procedure has been investigated by means of several techniques, including thermogravimetry, Raman spectroscopy, transmission electron microscopy, and cyclic voltammetry. A functionalization of about 1 lysozyme molecule every 4000 carbon atoms is obtained. The modified lysozyme-CNTs nanocomposite shows a significant increase of the antibacterial activity towards the Gram-positive S. aureus if compared with lysozyme in solution. Keywords: Nanomedicine, Carbon Nanotube, Lysozyme, Covalent Binding, Antibacterial Activity. 1. INTRODUCTION Carbon nanotubes (CNTs) as diagnostic or therapeutic tools in nanomedicine are arousing a widespread and increasing interest. 1–4 Carbon nanotube protein conjugates have been recently considered both for the delivery of therapeutics and for their application as electrochemical biosensors. 5 In particular, peptides were known to have a particular affinity towards CNTs, allowing their dis- persion in water by increasing their solubility in aque- ous medium. 6 The entrapment of proteins into the inner space of opened CNTs has been also reported. 7 Gener- ally speaking, both covalent and non-covalent binding to CNTs can be envisaged to link a protein onto the CNT surface. However, the non-covalent interaction between a polypeptide and the CNTs can lead to marked modifi- cations of the former due to adsorption interactions. As an example, either -chymotrypsin or soybean peroxidase adsorbed onto CNTs showed a partial or total loose of their native activity, as evidenced by FT-IR spectroscopy analysis. 8 As from the above, it is apparent that the protein ∗ Author to whom correspondence should be addressed. structure and functions are strongly influenced by the car- bon nanotube environment, and the bioactivity modifica- tion could be ascribed to a conformational change caused by the peptides adsorption onto CNTs surface. 9 Concerning the covalent immobilization, some stud- ies were reported where peptides, when tethered by chemoselective ligation to CNTs, showed a conformational behaviour similar to that of the free peptide in solution. 10 However, insufficient data are available on the fate of enzy- matic activity of peptides linked to CNTs in an unselective fashion 11 and, in many cases, the covalent attachment was claimed but not satisfactory proven. 11 Lysozyme is a good model system to investigate the advantages related to the CNTs covalent function- alization. It is well known to kill Gram-positive bacte- ria, by hydrolyzing 1,4 O-glycosidic subunit among N -acetylmuramic acid and N-acetylglucosamine subunits of peptidoglycane, which is an essential component of bacterial. 12 In the recent years, lysozyme was used to disperse CNTs. 1314 The feasibility of biomimetic, large- scale antimicrobial coatings based on single-walled CNTs (SWCNTs) and lysozyme was recently demonstrated. 15 However, only one report on covalent binding with SWC- NTs has been reported, 16 whereas nothing appeared to date J. Nanosci. Nanotechnol. 2011, Vol. 11, No. xx 1533-4880/2011/11/001/007 doi:10.1166/jnn.2011.3758 1