Elsevier

Journal of Biotechnology

Volume 189, 10 November 2014, Pages 114-119
Journal of Biotechnology

Eco-friendly synthesis of shrimp egg-derived carbon dots for fluorescent bioimaging

https://doi.org/10.1016/j.jbiotec.2014.08.043Get rights and content

Highlights

  • We used Shrimp egg as a carbon source for the fabrication of fluorescent CDs.

  • AFM images show the SE-CDs are spherical and monodispersed on Si substrate.

  • SE-CDs exhibit good water solubility with no further surface modification required.

  • SE-CDs are highly biocompatible and efficiently taken up by cells.

Abstract

Developing synthetic methods to produce carbon dots (CDs) using natural biomass or other readily available carbon sources are currently being explored. We describe a simple and green synthetic method for preparing fluorescent CDs by water extraction from heat-treated shrimp eggs (SE-CDs). The SE-CDs appeared spherical with an average size of 3.25 ± 1.06 nm. Elemental analysis indicate that the SE-CDs have functional groups such as C–OH, C–O–C, Cdouble bondO, and C–H on the surface which give rise to a series of emissive traps between π–π* states. SE-CDs also showed a broad emission range with excellent quantum yield of 18.5 ± 2.6%. In addition, when compared with commonly used traditional CdSe and CdTe nanocrystals, SE-CDs were bio-tolerable to cell at high doses (200 μg ml−1) in MTT assay. Thus, SE-CDs are very promising alternatives to semiconductor-based quantum dots for in vitro and in vivo bioimaging applications.

Introduction

Previously, photoluminescent semiconductor-based quantum dots (QDs) have shown several inherent advantages over conventional fluorophore dyes, such as high absorption cross-section, long fluorescent lifetimes, tunable emission peaks and negligible photodegradation, making them useful fluorescent tools for bio-applications (Clapp et al., 2005, Lim et al., 2003). However, the QD surface is typically hydrophobic and thus incompatible with aqueous and biological environments. Consequently, QDs must be subjected to secondary chemical processing to modify the outer surface to make them water soluble (Clapp et al., 2005). This serves the dual purpose of providing a mechanism for adding specific chemical functionalities as well as facilitating the removal of the toxic QD core materials from the environment. As such, functionalized, water-soluble QDs could be well-dispersed in a variety of aqueous buffers, and conjugated with biomolecules (Clapp et al., 2003, Clapp et al., 2005). However, the applied coating on QDs increases their size as compared to standard fluorophores which may be a hindrance in biological systems (Walling et al., 2009). Furthermore, their inherent toxicity makes them non-ideal for biological applications. Thus, substantial effort has gone into developing a smaller, hydrophilic and non-toxic substitute.

Carbon dots (CDs) are carbonaceous nanoparticles that exhibit physical and optical properties analogous to conventional QDs and silicon nanoparticles (Lin et al., 2013, Zhang et al., 2011). Compared to QDs, fluorescent CDs are superior in terms of their aqueous solubility, small size (<10 nm), intense brightness, high photostability, low cytotoxicity, and good biocompatibility (Wang et al., 2011). Additionally, CDs are also relatively easy to functionalize, low-cost, and applicable in large scale (Wang et al., 2013). Thus, photoluminescent CDs are promising alternatives to semiconductor QDs for applications such as bioimaging, biosensing, photocatalysis, and drug delivery (Baker and Baker, 2010, Hsu et al., 2012, Hu et al., 2009, Jaiswal et al., 2012, Zhang et al., 2012). Previous studies have reported on the preparation of CDs using natural biomass or other readily available carbon source materials. Liu et al. (2007) fabricated photoluminescent carbon nanoparticles (CNPs) from the combustion soot of candles. Using plant-based biomass, Krysmann et al. (2012) reported an environmentally benign approach to synthesize CNPs based on the pyrolytic decomposition of shredded grass. Hsu et al. (2012) developed a synthetic route for preparing CDs using coffee grounds as the starting material without strong acid or surface passivation. Other related studies of biomass-derived CNPs have been reported, using soy milk (Liu et al., 2007), pomelo peel (Qian et al., 2006), and orange juice (Sahu et al., 2012), as the starting material via a variety of processing methods.

Biomass is an abundant and important feedstock for the production of a range of carbonaceous materials including nanoparticles (Krysmann et al., 2012), nanotubes (Shinde and Pillai, 2012), and nanofibers (Qian et al., 2006). However, to the best of our knowledge, no examples have been published where readily-abundant ocean biomass was used as the starting material for fabrication of CNPs. Herein, we report a facile method for fabrication of strongly photoluminescent CDs by water extraction from heat-treated shrimp eggs (SE-CDs). We also demonstrate that the CDs prepared by this method are biocompatible, biodegradable, and are able to permeate a live cell membrane, thereby making them favourable candidates for in vivo biomedical imaging applications.

Section snippets

SE-CDs (carbon dots) synthesis

Carbon dots were fabricated by placing 5 g of fresh shrimp eggs in a clean glass dish, heating in an oven at 180 °C for 25 min, and then cooling the sample to room temperature. This process results in the conversion of the shrimp egg constituents to form carbon dots. Subsequently, cold-water extraction and ultrasonic extraction methods were used to prepare a stock solution of carbon dots from the heat-treated shrimp eggs. The procedures were performed as follows: 0.1 g treated shrimp eggs were

Structural characterization of SE-CDs

The as-prepared SE-CDs were deposited on a Si substrate for characterization by atomic force microscopy (AFM). Fig. 1a shows the SE-CDs appear spherical and are monodispersed on the Si surface. The size distribution histogram in Fig. 1a (inset) was calculated by measuring the height of 114 nanoparticles and shows that the SE-CDs have an average size of 3.25 ± 1.06 nm. High-resolution transmission electron microscopy (HRTEM) was used to further confirm the SE-CD particle size and nanocomposite

Conclusion

We have demonstrated a green and accessible synthetic approach for fabricating fluorescently stable CDs using shrimp eggs as a readily available natural carbon-source precursor. In contrast to most chemical approaches, the SE-CDs exhibit excellent luminescence, as well as good water solubility without any strong acid or surface passivation reagent modification. Based on previous studies (Hsu et al., 2012), CDs prepared from hydrophilic compounds have greater QY than hydrophobic ones. In

Acknowledgments

The authors would like to thank the Center for Stem Cell Research, Kaohsiung Medical University, Kaohsiung, Taiwan (KMU-TP103G01, KMU-TP103G03, KMU-TP103G04, and KMU-TP103G05), the Ministry of Science and Technology (NSC 101-2113-M-110-013-MY3) of Taiwan and the National Sun Yat-sen University Center for Nanoscience and Nanotechnology for financial support of this work. Prof. Hsieh also thanks Dr. Ming-Hong Tai for providing the fluorescence microscope and SK-Hep-1 cell line, and Dr. David Beck

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