One-step hydrothermal approach to fabricate carbon dots from apple juice for imaging of mycobacterium and fungal cells

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Highlights

  • The CDs are synthesized by hydrothermal method using apple juice as a precursor.

  • The CDs exhibited bright blue fluorescence at excitation wavelength of 365 nm.

  • The prepared CDs are in spherical and monodisperse with an average size of 4.5 ± 1.0 nm.

  • The CDs used as fluorescent probes for M. tuberculosis, P. aeruginosa and M. oryzae cells.

  • The CDs show biocompatibility and efficiently taken by the cells.

Abstract

A facile green hydrothermal method was developed for the preparation of fluorescent carbon dots (CDs) using apple juice as a raw material. The synthesized fluorescent CDs were characterized by UV–vis, fluorescence, Fourier transform infrared (FT-IR), dynamic light scattering (DLS), high resolution transmission electron microscopic (HR-TEM), life-time measurement and laser scanning confocal microscopic techniques. The CDs showed bright blue emission under UV-light (λex = 365 nm). The CDs were used as alternative biocompatible fluorescent probes for imaging of bacteria (Mycobacterium tuberculosis and Pseudomonas aeruginosa) and fungal (Magnaporthe oryzae). It was shown that the prepared CDs had no toxic effect on the both cells lines (bacteria and fungal), indicating that the CDs did not inhibit growth of bacterial and fungal cells, which confirms that the CDs exhibit good biocompatibility.

Introduction

In the ever-expanding field of nanomaterial research, the CDs have attracted much interest over the last decade due to their remarkable novel properties such as biocompatibility, low toxicity, easy functionalization, chemical inertness, photostability, and valuable photoluminescence [1], [2]. In recent years, the CDs have proved to be as potential eco-friendly fluorescent probes for visualizing structural or functional images of living systems down to the molecular level. The advent of new optical properties of CDs has made the research on the development of various “smart” nanosystems such as bioimaging [3], [4], [5], [6], biochemical assays [7], [8], drug delivery [9], photocatalysis [10] and light-emitting devices [11], [12]. As a result, much efforts have been focused on the development of various synthetic approaches for the preparation of fluorescent CDs including laser ablation [13], [14], arc discharge [15], pyrolysis [16], oxidation [17], electrochemical exfoliation [18], hydrothermal treatment [19], plasma treatment [20], microwave irradiation [21], [22], [23], [24] and microwave/ultrasonic passivation [25], [26]. On the other hand, even though the above methods were successfully applied to prepare CDs, unfortunately they require tedious process, sophisticated instrumental set-up, limited spectral efficiency, and low product yield. In view of green chemistry approach, there is an urgent need to establish a new green synthetic approach for the preparation of fluorescent CDs and their applications in bioimaging.

Over the past few years, hydrothermal carbonization has proved as an eco-friendly, traditional and soft chemical route for the preparation of CDs in aqueous media, which produces highly efficient fluorescent probes for recognizing various chemical species and cells in vitro and in vivo [27]. Hydrothermal synthesis of CDs using various renewal carbon sources has provided great advancement over existing physical methods (laser ablation, arc discharge and plasma treatment), which is due to its simplicity and production of CDs with good quantum yield. In hydrothermal method, CDs were prepared by using either low-cost or biowaste materials as raw materials. For example, CDs were successfully prepared by using various renewable resources as precursors including chitosan [28], coffee grounds [29], watermelon peel [30], pomelo peel [31], Trapa bispinosa peel [32], orange juice [33], gelatin [34], and low-cost organic chemicals [35], [36]. It can be noticed that the CDs were successfully generated using bio-waste/plant materials as raw materials, which avoids the use of costly/toxic chemicals or large amounts of chemicals and complicated post-treatment processes.

Furthermore, Li's and Zhang's groups [37] developed a new strategy for the preparation of two types of CDs with either excitation-independent blue emission or distinctive excitation-dependent full-color emissions using chloroform and diethylamine as precursors. Tian and co-workers [38] developed a CDs-based fluorescence detection method for sensing of reactive oxygen species in biological tissues. A novel and large-scale strategy was developed for the preparation of N-doped CDs with high yield by pyrolyzing ethanolamine as a precursor [39]. Dong et al. [40] described one-step microwave method for large-scale fabrication of water dispersible fluorescent CDs using citric acid and ethylenediamine as precursors for bioimaging of cells and sensing of Hg2+ ion. Shao's group [41] prepared N-doped graphite-like phase CDs for multicolor real-time imaging of cancer cells. Wang's and Chen's teams [42] reported a simple green synthetic method for the fabrication of fluorescent CDs using hair as a raw material. Bendicho et al. [43] developed in situ ultrasound-assisted synthetic method for preparation of CDs using fructose as a carbon source for the detection of methylmercury. Recently, various natural precursors such as konjac flour [44], bamboo leaves [45], sugar cane juice [46] and milk [47] have been used as carbon sources for the preparation of fluorescent CDs and acted as probes for imaging of cells and sensing of inorganic species. Furthermore, Jun et al. [48] illustrated the use of apple juice as a carbon source for the preparation of fluorescent CDs and used as probes for sensing of Hg2+ ion in environmental water samples. Abdollahi's group [49] also synthesized CDs from fruit juice and used as a fluorescent probe for the sensitive and selective detection of Hg2+ ion with a detection limit of 14 nM. These reports illustrated that the selection of precursors play a key role to modify the surface chemistry of CDs with multiple functional groups for sensing and cellular imaging of target analytes in biocomplex samples. To date, no reports are available for imaging of bacteria (Mycobacterium tuberculosis, Pseudomonas aeruginosa) and fungal (Magnaporthe oryzae) cells using apple juice derived CDs as imaging probes.

Herein, we report a simple hydrothermal method for the preparation of fluorescent CDs using apple juice as a raw material. The prepared CDs are highly water-soluble, nano-sized (4.5 ± 1.0 nm) and emitted bright blue fluorescence. The surface chemistry, size, and morphology of CDs were characterized by FT-IR, DLS and HR-TEM. The multicolor CDs are quite stable against photo-bleaching as compared with organic dyes and used as fluorescent probes for imaging of bacteria (P. aeruginosa and M. tuberculosis) and fungal (M. oryzae) cells (Scheme 1).

Section snippets

Chemicals

Malus domestica (apple) was purchased (Rupees 50/- for 500 g) from the local market of Surat, India and used as a precursor. Dichloromethane, sodium chloride, glucose, glycerol and dimethyl sulfoxide were obtained from Merck Ltd., India. All chemicals were of analytical grade and used without further purification. Milli-Q-purified water was used for sample preparations.

Synthesis of carbon dots from apple juice

The fluorescent CDs were synthesized via hydrothermal carbonization method using apple juice as a carbon source [48]. Briefly,

Characteristics of CDs

The CDs were successfully synthesized by the hydrothermal route using apple juice as a raw material since apple contains a wide variety of organic molecules (carbohydrates, polyphenols and volatile organic compounds). To convert these organic molecules into carbon nanostructures, hydrothermal carbonization was carried out at 150 °C for 12 h. As shown in Scheme 1, the CDs show strong blue emission under ultraviolet illumination at 302 and 365 nm, confirming that the formation of fluorescent CDs.

Conclusions

In the present work, water dispersible fluorescent CDs were synthesized by the hydrothermal carbonization using apple juice as a precursor without further chemical modification. The surfaces of CDs were functionalized with multifunctional groups (hydroxy, keto, carboxylic acid, and amino) and showed good QY (4.27%). Moreover, the spectroscopic (UV–vis, fluorescence, FT-IR, and DLS) and microscopic (HR-TEM and XRD) results indicated that the CDs have small oxygenous turbostratic and graphitic

Acknowledgements

Authors thank Prof. Z. V. P. Murthy, Chemical Engineering Department, S. V. National Institute of Technology, Surat for providing DLS instrument facility. Authors also thank Department of Science and Technology, Government of India for providing Maya Pro 2000 spectrophotometer (Ocean Optics, USA) under the Fast-Track Young Scientist Scheme (Ref. No: SR/FT/CS-54/2010). Mr. V. N. Mehta greatly acknowledge Department of Science and Technology, Government of India (Ref. No: SB/ITS-Y/02901/2014-15)

Vaibhavkumar N. Mehta completed his Master degree in Nanoscience and Nanotechnology from Sardar Patel University, Gujarat. Currently he is doing his Ph.D. at S. V. National Institute of Technology, Surat. His research interest is functionalization of nanoparticles for colorimetric sensing of biomolecules and metal ions.

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    Vaibhavkumar N. Mehta completed his Master degree in Nanoscience and Nanotechnology from Sardar Patel University, Gujarat. Currently he is doing his Ph.D. at S. V. National Institute of Technology, Surat. His research interest is functionalization of nanoparticles for colorimetric sensing of biomolecules and metal ions.

    Sanjay Jha completed his Ph.D in Microbiology from M.S. University, Baroda, Gujarat. Currently, he is working as Associate Professor, Gujarat Agricultural Biotechnology Institute, Navsari Agricultural University, Surat, India. He is interested in nanomaterials interactions with cells and their confirmations by microscopic tools.

    Hirakendu Basu is working as Scientist, Analytical Chemistry Division, Bhabha Atomic Research Center, India. His research area is on the development of analytical methods for the analysis of trace metal ions in different environmental matrices.

    Rakesh Kumar Singhal passed BARC Training School (32nd batch) from Chemistry stream. He has obtained his master degree in chemistry from I.I.T. Delhi and Ph.D. (Chemistry) from Mumbai University in 1996. He is the Head, Analytical Spectroscopy Section of Analytical Chemistry Division of Bhabha Atomic Research Center, India is responsible for development of analytical spectroscopic techniques for the measurement of traces of various elements in different environmental matrices and samples originating from different chemical processes. He has authored more than 150 publications in International & national Journals, Symposium and Book Chapters, in the field of environmental & radioanalytical chemistry.

    Suresh Kumar Kailasa completed his Ph.D. in Chemistry from S. V. University, Tirupati in 2005. After completing his two postdoctoral fellowships at South Korea and at National Sun Yat-Sen University, Taiwan he joined as a faculty at S. V. National Institute of Technology, Surat in 2009. He received Young Scientist Award from Taiwan Mass Spectrometry Society in 2013. His interest includes nanomaterials integration in MALDI-MS and development of nanomaterials-based colorimetric sensors for biomolecules, drugs and metal ions.

    This work was orally presented at 1st International Caparica Conference on Chromogenic and Emissive Materials, 8th–10th September 2014, Lisbon, Portugal.

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