Solid-state synthesis of self-functional carbon quantum dots for detection of bacteria and tumor cells

https://doi.org/10.1016/j.snb.2016.01.062Get rights and content

Highlights

  • Development of a simple solid-state synthesis for preparation of biofunctional fluorescent carbon quantum dots (CQDs).

  • Mannose (Man) and folic acid (FA) were self-functionalized to CQDs.

  • Man–CQDs can be used for selective labeling of E. coli.

  • FA–CQDs are highly selective for folate receptor-overexpressing tumor cells.

Abstract

We have developed a simple solid-state synthesis procedure to functionalize fluorescent carbon quantum dots (CQDs), using ammonium citrate as a carbon source and appropriate molecules as recognition ligands. Mannose and folic acid were used to modify CQDs to selectively label Escherichia coli (E. coli) and tumor cells, respectively. First, fluorescent-core CQDs (approximate size: 3 nm) were synthesized through carbonization of ammonium citrate via dry heating. In the second step, CQDs were heated with mannose and folic acid to prepare mannose-functionalized CQDs (Man–CQDs) and folic acid-functionalized CQDs (FA–CQDs), respectively, through a dehydration reaction in the solid state. Solid-state synthesis of the self-functional CQDs is achievable without a coupling agent. We optimized the labeling efficiencies of self-functional Man–CQD and FA–CQD to cells by controlling the ratio of mannose or folic acid to CQDs, as well as the reaction temperature during synthesis. The solid-state synthesized Man–CQDs and FA–CQDs exhibited excitation-dependent fluorescence with excitation and emission maxima of 365 and 450 nm, respectively, and a fluorescence quantum yield of approximately 9%. Man–CQDs can be used for selective labeling of E. coli and detection at concentrations as low as 100 colony forming units mL−1 in real samples (e.g., drinking water, apple juice, urine). Furthermore FA–CQDs are highly selective for labeling of folate receptor-overexpressing tumor cells. The synthesis of self-functional CQDs is simple, cost effective, and easily scaled up, and can be extended to the synthesis of various functional carbon nanomaterials, such as graphene oxide nanosheets, carbon nanotubes, fullerene nanoparticles and carbon nanodiamonds, with different ligands for other biolabeling applications and targeted therapies.

Introduction

Carbon quantum dots (CQDs), a new type of fluorescent carbon nanomaterial, are quasi-spherical nanoparticles with sizes ranging from 3 to 10 nm, and are comprised mainly of sp2 and sp3 hybridized carbon atoms [1], [2], [3]. CQDs have become attractive labeling agents because of their controllable emission wavelengths, high quantum yields (QYs), excellent photobleaching resistance, and good biocompatibility [4], [5], [6], [7]. Accordingly, CQDs have been widely used to label bacteria, fungi, and tumor cells [8], [9], [10], [11], [12], [13], [14], [15]. However, the complex processes of passivation of CQDs and anchoring of recognition molecules using toxic and/or expensive coupling agents and tedious separations are indispensable in the functionalization of CQDs for selective targeting [16], [17], [18], [19], [20], [21], [22], [23], [24]. Moreover, decrease in the fluorescence QYs and aqueous-phase dispersibility of CQDs usually arise following complicated modifications and separations. Thus, the development of simple synthesis methods for the biofunctionalization of CQDs with high QYs and dispersibility remains a significant challenge.

Herein, we report a simple synthesis approach comprising a two-step dry heating reaction for the preparation of self-functional CQDs (Scheme 1). In this process, the cores of fluorescent CQDs (size: ca. 3 nm) were synthesized by heating solid-state ammonium citrate. The functional ligand shells were formed by anchoring mannose or folic acid molecules to the CQD surfaces through a dehydration reaction under dry heating. Both the prepared mannose-functionalized CQDs (Man–CQDs) and folic acid-functionalized CQDs (FA–CQDs) exhibited bright blue fluorescence and high labeling efficiency for their respective targets, Escherichia coli (E. coli) and folate receptor-overexpressing tumor cells. Compared with the one-step synthesized Man–CQDs described in our previous report [25], the two-step synthesized Man–CQDs exhibited higher efficiency in the labeling of E. coli. In the one-step method, both the formation of CQDs and the functionalization took place simultaneously, and the mechanism of formation of mannose-functionalized CQDs is ambiguous. However, in the present work we demonstrated the formation of CQDs took place in first step followed by its functionalization process in second step. In this case, it is logical to substantiate that the CQDs and functional molecules formed a core–shell structure. Furthermore, the ligand density (mannose or folic acid) on the CQDs is controllable in the two-step synthesis. These self-functional CQDs can be synthesized from the solid state without any coupling agents. In addition, the fluorescent properties and stability of CQDs did not change after functionalization.

Section snippets

Synthesis of biofunctional carbon quantum dots

First, CQDs were synthesized using a simple heating method. Briefly, 2 g of ammonium citrate powder was placed in a 20 mL beaker and heated in an oven at 180 °C for 2 h to yield a black residue. The formed CQDs were cooled to room temperature, ground to a fine powder, and stored in a dry cabinet for future use. To synthesize Man–CQDs, a crucible containing prepared CQDs (0.05 g) and mannose (0.005 g) was placed in an oven and heated at 180 °C for 2 h. The resulting black residue was cooled to room

Preparation of Man–CQDs

First, the core CQDs were prepared by heating dry-state ammonium citrate (2 g) at 180 °C for 2 h [26]. In the second step, finely ground CQDs (0.05 g) and mannose (0.005 g) were well-mixed and heated at 180 °C for another 2 h. The synthesized brown Man–CQD product was then dispersed using NaOH (0.1 M, 5 mL; inset in Fig. 1B) for a yield of approximately 70% (7.5 mg mL−1). The transmission electron microscopy (TEM) images (Fig. 1A) reveal that the particle sizes of as-prepared of CQDs (3.1 ± 1.5 nm;

Conclusions

We have demonstrated a simple dry-heating method for self-functionalizing CQDs with different molecules, thus facilitating biolabeling. The fluorescent properties of CQDs, Man–CQDs, and FA–CQDs did not differ greatly, indicating that functionalization did not affect the core structure of the CQD. The dry-heating method readily facilitated the anchoring of functional ligands to the CQDs via a dehydration coupling reaction. The synthesized Man–CQDs and FA–CQDs exhibited highly efficient cell

Acknowledgments

This study was supported by the Ministry of Science and Technology of Taiwan under the contracts 104-2628-M-019-001-MY3, 103-2627-M-007-002-MY3, and 102-2113-M-019-001-MY3. We appreciate the assistance of Ms. Ya-Yun Yang and Ms. Ching-Yen Lin from the Instrument Center of National Taiwan University (NTU) with TEM measurements.

Irving Po-Jung Lai is currently doing his Doctoral studies at the Department of Bioscience and Biotechnology, National Taiwan Ocean University, Taiwan. His research interest is synthesis of carbon-based nanomaterials for the biolabelling.

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    Irving Po-Jung Lai is currently doing his Doctoral studies at the Department of Bioscience and Biotechnology, National Taiwan Ocean University, Taiwan. His research interest is synthesis of carbon-based nanomaterials for the biolabelling.

    Scott G. Harroun is a Research Assistant in the laboratory of Dr. Huan-Tsung Chang in the Department of Chemistry at National Taiwan University. He previously earned his B.Sc. and M.Sc. degrees under the supervision of Dr. Christa Brosseau in the Department of Chemistry, Saint Mary's University, Canada. He will return to Canada in September for his doctoral studies in chemistry at the Université de Montréal. His research interests are SERS and electrochemistry for biosensing applications.

    Shiow-Yi Chen is an Assistant Professor of the Department of Bioscience and Biotechnology, National Taiwan Ocean University, Taiwan. Her research work is mainly focused on development of functional nanomaterials in tumor therapy.

    Binesh Unnikrishnan is currently working as Postdoctoral Fellow at the Department of Bioscience and Biotechnology, National Taiwan Ocean University, Taiwan. His research interests include synthesis of nanomaterials for biosensor and energy devise applications.

    Yu-Jia Li is currently doing his Doctoral studies at the Department of Bioscience and Biotechnology, National Taiwan Ocean University, Taiwan. Her research interest is synthesis of antibacterial nanomaterials.

    Chih-Ching Huang is a Professor of the Department of Bioscience and Biotechnology, National Taiwan Ocean University, Taiwan. He received his Ph.D. degree in Analytical Chemistry from the Department of Chemistry with Dr. Huan-Tsung Chang, National Taiwan University, Taiwan (2004). He worked as Postdoctoral fellow at the Department of Chemistry, National Taiwan University (2006–2008). In 2008, he joined the Department of Bioscience and Biotechnology, National Taiwan Ocean University, Taiwan. His research work is mainly focused on development of nanosensors and nanodrugs.

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