Blue-Emitting Copper Nanoclusters Synthesized in the Presence of Lysozyme as Candidates for Cell Labeling Rama Ghosh, † Amaresh Kumar Sahoo, ‡ Siddhartha Sankar Ghosh, ‡,§ Anumita Paul,* ,† and Arun Chattopadhyay* ,†,‡ † Department of Chemistry, ‡ Centre for Nanotechnology, and § Department of Biotechnology, Indian Institute of Technology Guwahati, Guwahati-781039, India * S Supporting Information ABSTRACT: Highly fluorescent copper nanoclusters (Cu NCs) have been synthesized using single-step reduction of copper sulfate by hydrazine in the presence of lysozyme. The fluorescence quantum yield was measured to be as high as 18%. The emission was also found to be dependent on the excitation wavelength. Mass spectrometric analyses indicated the presence of species corresponding to Cu 2 to Cu 9 . Transmission electron microscopic analyses indicated the formation of agglomerated particles of average diameter of 2.3 nm, which were constituted of smaller particles of average diameter of 0.96 nm. They were found to be stable between pH 4 and 10 and in addition having excellent chemical and photostability. The noncytotoxic NCs were used to success- fully label cervical cancer HeLa cells. KEYWORDS: copper nanoclusters, fluorescence, HeLa cells, photostability, cytotoxicity ■ INTRODUCTION Stabilizing nanoclusters (NCs) of noble metal remains the primary challenge in their syntheses and applications. It has been proposed that these highly luminescent and photostable NCs could substitute toxic quantum dots in bioimaging and biolabeling. 1,2 In this regard, owing to their redox properties, NCs of gold (Au), and to some extent those of silver (Ag), have been stabilized with considerable success in comparison to those of copper (Cu). For example, recently, syntheses of luminescent Au and Ag NCs have been reported using dendrimers, 3 poly(acrylic acid) polymer, 4,5 DNA, 6,7 and protein 8 as stabilizers. Among the proteins, bovine serum albumin, 8-10 lacto transferrin, 11 lysozyme, 12,13 insulin, 14 horse- radish peroxidase, 15 and pepsin 16 have been preferred as stabilizers. It is important to mention here that the proposed use of these clusters in a biological environment demands their stability in an aqueous medium. This has remained a challenge for the synthesis of NCs of Cu. The ease of oxidation of Cu (E 0 , 0.34 V), in comparison to that of Ag (E 0 , 0.80 V) and Au (E 0 , 1.50 V), has limited progress in the development of synthetic methods, especially in an aqueous medium. A recent report indicates that Cu NCs of less than 3 nm, synthesized using a polyol method under a N 2 atmosphere, were stable following their redispersion in an aqueous medium. 17 On the other hand, the possibility of the synthesis of small Cu NCs in an aqueous medium electrochemically has also been demonstrated. 18 Interestingly, DNA-hosted Cu NCs, synthesized in the presence of ascorbic acid, have been used for the identification of single nucleotide polymorphism. 19 However, there is a need for the development of aqueous-based synthetic methods for versatile use, especially in biological applications. Cu is an important trace element, being present in the human body as an essential catalytic cofactor in redox-active enzymes such as cytochrome c oxidase and lysyl oxidase. The permissible intake of Cu for an adult is 0.6-1.6 mg/day. 20 The ease of sequestration of Cu by a natural bodily mechanism and the availability of commercial chelating agents also make its use relatively friendly in human subjects over those of Au and Ag, especially at low concentrations. 21,22 Herein we report the synthesis of highly fluorescent, blue- emitting Cu NCs by chemical reduction of CuSO 4 in an aqueous medium, in the presence of lysozyme. Lysozyme, a 14.3 kDa protein, has 129 amino acid residues including 8 cysteine residues. The antimicrobial protein is biocompatible and has been a favorite as a stabilizer of Au NCs. 13 The so- synthesized protein-stabilized Cu NCs, with wavelength- tunable emission, were stable in an aqueous medium under ambient conditions; they could easily be isolated and used for labeling cervical cancer HeLa cells. Cell viability studies indicated their noncytotoxic nature, making the NCs ideal for biological applications. The essential steps of the synthesis are depicted in Scheme 1. Received: September 23, 2013 Accepted: February 26, 2014 Published: February 26, 2014 Research Article www.acsami.org © 2014 American Chemical Society 3822 dx.doi.org/10.1021/am500040t | ACS Appl. Mater. Interfaces 2014, 6, 3822-3828