Biosensors and Bioelectronics 60 (2014) 292–298
Contents lists available at ScienceDirect
Biosensors and Bioelectronics journal homepage: www.elsevier.com/locate/bios
Novel and green synthesis of high-fluorescent carbon dots originated from honey for sensing and imaging Xiaoming Yang n,1, Yan Zhuo 1, Shanshan Zhu, Yawen Luo, Yuanjiao Feng, Yao Dou College of Pharmaceutical Sciences, Southwest University, Chongqing 400716, China
art ic l e i nf o
a b s t r a c t
Article history: Received 12 February 2014 Received in revised form 10 April 2014 Accepted 22 April 2014 Available online 30 April 2014
An innovative and green strategy to synthesize carbon dots (CDs) with a quantum yield (QY) of nearly 19.8% has been successfully established for the first time. Subsequently, the possible fluorescence (FL) mechanism was elucidated by fluorescence, UV–vis, high resolution transmission electron microscope (HR-TEM), Fourier transform infrared spectroscopy (FTIR) and X-ray photoelectron spectroscopy (XPS) analyses. Significantly, not only the precursor of CDs and whole synthesis procedure was green, but also the CDs obtained here exhibited various advantages including high fluorescent QY, excellent photostability, non-toxicity and satisfactory stability. Additionally, the CDs were employed for assaying Fe3 þ based on direct interactions between Fe3 þ and –COOH, –OH and –NH2 of CDs, resulting in aggregations that facilitate to quench their fluorescence. The decrease of fluorescence intensity permitted detections of Fe3 þ in a linear range of 5.0 10 9–1.0 10 4 mol/L, with a detection limit of 1.7 10 9 mol/L at a signal-to-noise ratio of 3, suggesting a promising assay for Fe3 þ . Eventually, the CDs were applied for cell imaging and coding, demonstrating their potential towards diverse applications. & 2014 Elsevier B.V. All rights reserved.
Keywords: Carbon dots Green synthesis Sensing Bioimaging
1. Introduction Carbon nanomaterials, mainly including carbon nanotubes, fullerenes graphene, and carbon nanofilms, have been playing critical roles in various fields such as material science (Prato, 1997), biochemistry and biomedicine (Yang et al., 2009). Recently, carbon dots (CDs), emerging as a new class of fluorescent nanomaterials, have attracted considerable interest. Based on carbon skeleton structure, carbon dots usually exist in the size of less 10 nm, showing excellent properties such as ease of preparation (Jia et al., 2012; Liu et al., 2007), satisfactory fluorescent performance, low cytotoxicity and biocompatibility (Baker and Baker, 2010) owing to their specific nanometer dimension. Thus, CDs have been considered as a satisfactory candidate for biosensing (Wang et al., 2010), catalysis (Cao et al., 2011), and imaging (Wei et al., 2012; Zhao et al., 2011; Zhou et al., 2012). In the past few years, CDs were generally synthesized by two major methods. One way was known as top down, consisting of electrochemical oxidation (Zhao et al., 2008; Zhou et al., 2007), acidic oxidation (Dong et al., 2010), arc discharge (Xu et al., 2004) and laser ablation (Hu et al., 2009). Hydrothermal (Zhu et al., 2012), microwave (Salinas-Castillo et al., 2013) and ultrasonic (Zhuo et al., 2012), serving as another way
n
Corresponding author. Tel./fax: þ 86 23 68251225. E-mail addresses: [email protected], [email protected] (X. Yang). 1 Both authors contributed equally to this work.
http://dx.doi.org/10.1016/j.bios.2014.04.046 0956-5663/& 2014 Elsevier B.V. All rights reserved.
defined as bottom up, have been normally applied to synthesize CDs. Nevertheless, these methods exhibited several drawbacks such as tedious steps, toxic reagents, special equipments, strong acid (alkali) or high temperature and high costs, leading to their limitations for applications (Sahu et al., 2012). Therefore, exploring new methods for synthesizing CDs are still desired. As being well known, Fe3 þ served as an essential component of heme groups and one of abundant transition metals in human biological systems (Von Drygalski and Adamson, 2013). Currently, there exist various techniques for quantification of Fe3 þ including flame atomic absorption spectroscopy (Ajlec and Stupar, 1989), inductively coupled plasma mass spectrometry (Huang and Lin, 2001), atomic absorption/emission spectrophotometry (Wu et al., 2009), atomic fluorescence and UV spectrometry (Kok and Wild, 1960). However, most of these methods required sophisticated and expensive instrumentation and/or complicated sample preparation procedures and were time consuming (Lee et al., 2011; Wang et al., 2012; Xiang and Tong, 2006). In contrast, fluorescent sensors offered a simple, low-costly approach for assaying metal ions in biological and environmental samples. In this study, a simple, economical, and green method for preparing water-soluble CDs has been established with a quantum yield of 19.8%. Importantly, this strategy for producing CDs by one-step treatment of honey under low-temperature heating was reported for the first time (Fig. 1). In addition, both the precursor of CDs and the synthesis procedure are substantially environment friendly, leading to their biocompatibility and more extensive applications. Subsequently, we
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Fig. 1. Schematic illustration of synthesis of CDs and cells imaging (top) and coding (bottom).
applied the CDs for sensitive and selective detection of Fe3þ based on direct interactions between Fe3 þ and –COOH, –OH and –NH2 of CDs (Chinoporos, 1962; McIlwee et al., 2008; Moon et al., 2010), resulting in aggregations that facilitates the quenching of their fluorescence. Again, the practicability of this strategy has been validated by assaying Fe3þ in human blood samples. Meanwhile, the CDs were employed for cell imaging, coding and preparing fluorescent powder, suggesting their potential to broaden avenues for meaningful applications and commercial purpose.
2. Experimental 2.1. Chemicals and materials Honey was bought from Yonghui Supermarket (Chongqing, China). All the metal ions (Hg2 þ , Fe2 þ , Pb2 þ , Ag þ , Ca2 þ , Co2 þ , Mn2 þ , Sr2 þ , Zn2 þ , K þ , Na þ , Cu2 þ ) and trihydroxymethyl aminomethane (Tris) were obtained from Shanghai Sangon Biotechnonlogy Co., Ltd. (Shanghai, China). Disodium hydrogen phosphate (Na2HPO4) and sodium dihydrogen phosphate (NaH2PO4), 30% Hydrogen peroxide (30% H2O2), potassium dihydrogen phosphate (KH2PO4), citric acid and sodium citrate, glacial acetic acid (HAc), phosphoric acid (H3PO4), boric acid (H3BO3), sodium hydroxide NaOH), sodium chloride (NaCl), ammonium chloride (NH4Cl) were purchased from Dingguo Changsheng Biotechnology Co., Ltd. (Beijing, China). Ultrapure water, 18.25 MΩ cm, produced with an Aquapro AWL-0502-P ultrapure water system (Chongqing, China) was employed for all experiments.
2.3. Synthesis of CDs Basically, the green CDs were synthesized for the first time here. In brief, 2.0 g honey was added into 8 mL ultrapure water at the beginning. After stirring for mixing, this solution was introduced with 2 mL of 30% H2O2. Then the mixture was transferred into a 25 mL Teflon-lined stainless-steel autoclave. After heating at 100 1C for 2 h, the autoclave was cooled to room temperature, and this aqueous solution was adjusted to neutral with 2 M NaOH followed by filtered with 0.22 μm filter membrane to remove the larger product. Finally, the fluorescent carbon dots were collected by dialysis against deionized water though a dialysis membrane (1000MWCO) for 24 h. The powder of CDs was obtained by lyophilisation, and furhter dissolved in ultrapure water with the final concentration of 1 mg/mL. The CDs prepared here were stable for 3 months while stored in the dark at 4 1C. 2.4. Detection of Fe3 þ Firstly, 40 μL CDs (1 mg/mL), 40 μL Britton–Robinson (BR) buffer (50 mM, pH 6.0) and an appropriate volume of Fe3 þ working solution or sample solution were successively pipetted into a 1.5 mL vial. Subsequently, these solutions were diluted to 400 μL with Milli-Q purified water, and followed by vortex-mixed thoroughly. After reacting at 35 1C for 10 min, the mixtures were subjected to fluorescence measurements. Finally, interferences originated from other metal ions were investigated individually in the presence of CDs prepared here. 2.5. Preparation of blood samples
2.2. Instrumentation All fluorescence measurements were performed on a Hitachi F-7000 fluorescence spectrophotometer (Tokyo, Japan) with excitation slit set at 5 nm band pass and emission at 5 nm band pass in 1 cm 1 cm quartz cell. Meanwhile, UV/vis absorption spectra were recorded by a Shimadzu UV-2450 spectrophotometer (Tokyo, Japan). The high resolution transmission electron microscopy (HR-TEM) images were taken using a TECNAI G2 F20 microscope (FEI, America) at 200 kV. Elemental and functional groups analysis were obtained by an ESCALAB 250 X-ray photoelectron spectrometer and a Fourier transform infrared spectrometer (Tokyo, Japan), respectively. The quantum yields were obtained by using an Absolute PL quantum yield spectrometer C11347 (Hamamatsu, Japan). The powder of CDs obtained by lyophilisation in PiloFD84.3V (SIM, USA). Photographs of cells were taken with an Olympus fluorescence microscope 1 71 (Tokyo, Japan). A Fangzhong pHS-3C digital pH meter (Chengdu, China) was used to measure the pH values of the aqueous solutions and a vortex mixer QL-901 (Haimen, China) was used to blend the solution. The thermostatic water bath (DF-101s) was purchased from Gongyi Instrument Co., Ltd. (Henan, China).
Fresh human blood samples were originally collected from three healthy volunteers of Southwest University Hospital. In brief, 1 mL blood was initially dispensed in 5 mL of Red Blood Cell (RBC) lysis buffer, and incubated at room temperature for 15 min. Then, the lysed samples were centrifuged at 10,000 rpm for 10 min, and the supernatant were collected. Next, 0.2 mL 20% ascorbic acid was introduced into the above solutions to oxidize Fe2 þ to Fe3 þ for further analysis. Finally, the prepared samples were used for detections of Fe3 þ according to the general procedure without additional special treatment.
3. Results and discussion 3.1. Characterization of CDs To characterize this synthesized CDs, the maximum excitation and emission spectra of synthesized CDs were initially recorded as 338 nm and 420 nm (Fig. 2A) respectively, and the fluorescent properties of the CDs solution were subsequently investigated. The CDs aqueous solution emitted obvious blue fluorescence
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Fig. 2. (A) Fluorescence and UV–vis absorption spectra of CDs. Inset: photographs of CDs under daylight (I) and UV light (II). (B) Emission spectra of CDs for varying excitation wavelengths. (C)–(E) HR-TEM images of CDs.
Fig. 3. (A) FTIR of CDs; (B) XPS of CDs; (C) C 1s of XPS and (D) N 1s of XPS spectra of the CDs thus obtained.
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(photograph II) under UV light (365 nm) while appearing as completely colorless transparent under daylight (photograph I) in Fig. 2(A). Again, the UV–vis absorption spectrum showed a peak at 278 nm, owing to n–πn transition of CQO and π–πn transition of CQC (Fig. 2A). To address whether the synthesis of CDs showed excitation-dependent emission character or not, the emission peak shifted to longer wavelength and the fluorescence intensity decreased with the excitation wavelengths varying from 320 nm to 410 nm (Fig. 2B), suggesting the similar fluorescent properties. Besides, the as-synthesized CDs excited by 700 nm laser showed the maximum upconverted emission peak at 435 nm (Fig. S1), suggesting their infrared upconversion property, likely due to antiStokes photoluminescence (Ho et al., 2012; Mu et al., 2013). To further investigate the nanostructures of CDs, a high resolution transmission electron microscope (HR-TEM) was employed to directly observe the morphology and particle size distributions. As shown in Fig. 2(C) and (D), CDs obtained here existed as a majority population at the size of 2 nm and no aggregation emerged, depicting their satisfactory disparity. Meanwhile, the lattice spacing of 0.18 nm was accompanied with the [102] fact of graphitic carbon (Fig. 2E) (Wu et al., 2013; Zhu et al., 2012). Next, Fourier transform infrared spectroscopy (FTIR) and X-ray photoelectron spectroscopy (XPS) were applied to explore the surface groups, structure and components of these synthesized CDs in detail (Fig. 3). As revealed in Fig. 3(A), there existed O–H, N–H, C–H, CQN, CQO, CQC and C–O groups on the surface of CDs. Specifically, the absorption bands of O–H and N–H stretching vibrations appeared at 3380 cm 1 with C–H and CQN stretching vibrations at 2906 cm 1 and 2250 cm 1 respectively. Similarly, CQO and CQC stretching vibrations were at 1636 cm 1. Simultaneously, the peak at 1332 cm 1 was associated with C–H and N–H bending vibrations and the peak at 1071 cm 1 with C–O bending vibrations. To elucidate the components of the current
295
CDs, XPS survey spectra were performed. As shown in Fig. 3(B), three major peaks of C, O and N obviously emerged in the spectrum, indicating that CDs prepared here were mainly composed of C (44.24%), O (45.95%) and N. To fairly describe the peak of N, the amplified peak is shown in Fig. 3(D). Furthermore, there were three peaks displayed at 284.5 eV, 286.6 eV and 287.9 eV (Fig. 3C), demonstrating that there existed C–C, C–O and CQN/ CQO on the surface of CDs, which further confirmed the previous FTIR data. Overall, the FTIR data showed satisfactory agreement with that of XPS, intensively suggesting that the CDs produced here were equipped with characteristic functional groups including –COOH, –OH and –NH2, thereby facilitating their excellent water solubility without any chemical modification.
3.2. Stability of CDs Again, to assess the stability of CDs for bearing circumstances, various experiments were performed. As revealed (Fig. S2A–S2C), the fluorescent intensities of CDs scarcely exhibited variation along with varying concentrations of NaCl, pH and time, demonstrating the CDs outstanding stability, tolerance for pH and photobleaching. Moreover,
Table 1 Recoveries of Fe3 þ in human serum samples detected by the proposed method (n ¼6). Sample
Added (nM)
Measured (nM)
Recovery (%)
RSD (%)
Human blood
0 20 40 60
102.30 121.50 141.20 163.20
– 96.00 97.25 101.50
1.19 0.98 1.12 1.08
Fig. 4. (A) Fluorescence spectra of CDs in the absence (black) and presence (red) of 100 μM Fe3 þ . Inset: photographs; (B) effect of different metal ions on the fluorescence intensity of CDs. Inset: visual observation; (C) fluorescence spectra of CDs in the presence of various concentrations of Fe3 þ ; (D) the relationship between F0 F and Fe3 þ form 800 μM to 5 nM. Inset: plot of the fluorescent intensity decrease (F0 F) versus the logarithm of concentrations of Fe3 þ added. (For interpretation of the references to color in this figure legend, the reader is referred to the web version of this article.)
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Fig. S2(D) indicated that as-synthesized CDs could remain stable in the majority of organic solvents, as different solvents exhibited little effect on this CDs. Taken together, a brand-new type of CDs was successfully synthesized by a developed simple and green method. Considering that this CDs have been functioned with –COOH, –OH and –NH2 on the surfaces, we asked whether they could potentially serve as a
fluorescent probe to detect Fe3 þ or not (Chinoporos, 1962; McIlwee et al., 2008; Moon et al., 2010). 3.3. Optimization of analytical conditions Towards that purpose, Fe3þ was introduced into the CDs as prepared. Fig. 4(A) shows that fluorescence intensity of the CDs at
Fig. 5. Hep-2 cells in the absence (A) and presence of 1 mg/mL CDs incubated for 30 min (B) or 1 h (C); Hela cells in the absence (D) and presence of 1 mg/mL CDs for 30 min (E) or 1 h (F); images of powders including NaCl (left vial) and CDs (right vial) under daylight (G) and UV light (H); images of absorbent cotton (I) and filter paper (J) unstained (left) and stained (right) by this CDs under UV light.
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420 nm decreases to about 50%, when 100 mM Fe3þ is added. Simultaneously, the CDs solution shifted to light yellow under visible light (photograph III) and no fluorescence emission was observed under UV light (photograph IV), indicating that the CDs may be applied for detecting Fe3 þ . To elucidate the interaction between Fe3 þ and CDs, HR-TEM was used to directly observe this interaction. The sizes of CDs increased dramatically in the presence of 100 mM Fe3 þ (Fig. S3), suggesting aggregations occurred. To determine Fe3þ effectively, conditions including pH, reaction time, temperature and buffers were optimized. Fig. S4(A) shows that the fluorescence intensity of CDs is constant in the absence of Fe3 þ while the pH values vary from 3 to 11, suggesting that CDs are stable in acid and alkaline environment. Nevertheless, the fluorescence intensity of CDs greatly decreased in the presence of 100 mM Fe3 þ , and ΔF reached maximum at pH 6.0. Likewise, effects of reaction time (1 min, 10 min, 20 min, 30 min, 40 min, 50 min) and reaction temperature in the range of 30–60 1C were investigated towards 100 mM Fe3þ (Fig. S4B and S4C), claiming that ΔF of the CDs were independent of both the reaction time and temperature. Finally, effect of various buffers such as Phosphate Buffer Solution (PBS), Citrate–HCl, Phosphate Buffer (PB), BR and Acetic Acid–Sodium Acetate Buffer (HAc–NaAc) were tested, and ΔF of the CDs only in BR buffer reached maximum rather than that in other buffers (Fig. S4D). Considering the speed, easy-operation and selectivity of this assay, pH 6.0, 35 1C, 10 min and BR buffer served as the optimal conditions during the following experiments.
non-toxicity. Again, benefited from their high fluorescence and low toxicity, CDs were further employed for cell imaging. Specifically, HEp-2 and Hela cells were incubated in the absence (Fig. 5A and D) and presence of 1 mg/mL CDs, and there exhibited weak (30 min incubation) and obvious fluorescence (1 h incubation) originated from CDs in both cell lines (Fig. 5B, C, E, and F), suggesting that the CDs described here may play a role for cell imaging. In addition, as shown in Fig. 5(H), the powder of CDs obtained by lyophilisation (right vial) showed striking fluorescence under UV light compared with NaCl powder (left vial), whereas no difference was observed between CDs and NaCl powder under daylight (Fig. 5G). Significantly, the fluorescent powder has been preserved for more than 3 months without marked changes, indicating that it may provide the possibility for matching the requirement of commercial scale. Finally, we also applied the CDs as fluorescent dye for applications. Interestingly, both absorbent cotton and filter paper with staining (right) implied dramatic fluorescence under UV light compared with that without staining (left) in Fig. 5(I) and (J). Moreover, this CDs, serving as fluorescent ink, were used for producing various paintings (Fig. S6A–S6C), and the words written by the chalk stained with this CDs showed clear fluorescence (Fig. S6D). Hence, these photographs strongly demonstrated that these fluorescent CDs could serve for imaging and staining.
3.4. Selectivity of analytical strategy
4. Conclusions
Next, the selectivity of this analytical strategy was evaluated by testing the response to other metal ions (500 mM for each) under optimum conditions for the case of 10 mM Fe3 þ (Fig. 4B). Stock solutions of other metal ions (Hg2 þ , Fe2 þ , Pb2 þ , Ag þ , Ca2 þ , Co2 þ , Mn2 þ , Sr2 þ , Zn2 þ , K þ , Na þ , and Cu2 þ ) were prepared. Fig. 4 (B) shows that the fluorescence probe responds selectively toward the other metal ions, indicating the excellent selectivity of the CDs for Fe3 þ detection. Furthermore, the calibration curve was constructed under optimum conditions. The fluorescent intensity decrease (F0 F) versus the logarithmic plot of Fe3 þ concentration displayed a linear range from 5.0 10 9 mol/L to 1.0 10 4 mol/L (Fig. 4C). As shown in Fig. 4(D), (F0 F) was in a linear relationship along with concentrations of Fe3 þ , and the linear regression equation is (F0 F)¼1295.80 147.02 C3Feþ with a correlation coefficient of 0.9937 (n ¼5). Additionally, the relative standard deviation (RSD) was 3.1% and 1.7% for five repeated measurements of 5.0 10 9 and 1.0 10 4 mol/L Fe3 þ respectively, demonstrating an excellent precision of this fluorescent probe. The detection limit of Fe3 þ was 1.7 10 9 mol/L at a signal-to-noise ratio of 3. Overall, the results showed a highly sensitive and widely linear range method.
In summary, we have creatively synthesized CDs based on honey via a simple and green method for the first time. Moreover, these CDs were employed as a novel sensor for the quantification of Fe3 þ , which depended on Fe3 þ coordinated with –COOH, –OH and –NH2 of CDs (Chinoporos, 1962; McIlwee et al., 2008; Moon et al., 2010), thus leading to aggregations that quench their fluorescence. Significantly, this new CDs described here were applied for cell imaging and fluorescent staining, demonstrating many broaden potential avenues in bioimaging, biosensing and other purposes.
3.5. Detection of Fe3 þ in real samples For testing the practicality of this developed approach, standard recovery experiments were performed in human blood samples (Table 1). As listed, the recoveries of all samples were 96.00%, 97.25%, 101.50% respectively, and there were little inference from substrates of the blood, indicating the proposed method may broaden ways for practical detections of Fe3 þ in real samples. 3.6. Fantastic applications Ultimately, two types of crops were introduced to explore the toxicity of the CDs as-synthesized here. The seeds of green soya beans (Fig. S5A), and water spinach (Fig. S5B), incubated with CDs regularly grew in pace with time-variation compared with that without CDs incubations, revealing the CDs satisfactory
Acknowledgments We gratefully acknowledge financial support by National Natural Science Foundation of China (31100981), Research Fund for the Doctoral Program of Higher Education of China (20110182120014), Natural Science Foundation Project of CQ CSTC (cstc2013jcyjA10117), Fundamental Research Funds for the Central Universities (XDJK201 3B038), and Program for Innovative Research Team in University of Chongqing (2013).
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Contents lists available at ScienceDirect
Biosensors and Bioelectronics journal homepage: www.elsevier.com/locate/bios
Novel and green synthesis of high-fluorescent carbon dots originated from honey for sensing and imaging Xiaoming Yang n,1, Yan Zhuo 1, Shanshan Zhu, Yawen Luo, Yuanjiao Feng, Yao Dou College of Pharmaceutical Sciences, Southwest University, Chongqing 400716, China
art ic l e i nf o
a b s t r a c t
Article history: Received 12 February 2014 Received in revised form 10 April 2014 Accepted 22 April 2014 Available online 30 April 2014
An innovative and green strategy to synthesize carbon dots (CDs) with a quantum yield (QY) of nearly 19.8% has been successfully established for the first time. Subsequently, the possible fluorescence (FL) mechanism was elucidated by fluorescence, UV–vis, high resolution transmission electron microscope (HR-TEM), Fourier transform infrared spectroscopy (FTIR) and X-ray photoelectron spectroscopy (XPS) analyses. Significantly, not only the precursor of CDs and whole synthesis procedure was green, but also the CDs obtained here exhibited various advantages including high fluorescent QY, excellent photostability, non-toxicity and satisfactory stability. Additionally, the CDs were employed for assaying Fe3 þ based on direct interactions between Fe3 þ and –COOH, –OH and –NH2 of CDs, resulting in aggregations that facilitate to quench their fluorescence. The decrease of fluorescence intensity permitted detections of Fe3 þ in a linear range of 5.0 10 9–1.0 10 4 mol/L, with a detection limit of 1.7 10 9 mol/L at a signal-to-noise ratio of 3, suggesting a promising assay for Fe3 þ . Eventually, the CDs were applied for cell imaging and coding, demonstrating their potential towards diverse applications. & 2014 Elsevier B.V. All rights reserved.
Keywords: Carbon dots Green synthesis Sensing Bioimaging
1. Introduction Carbon nanomaterials, mainly including carbon nanotubes, fullerenes graphene, and carbon nanofilms, have been playing critical roles in various fields such as material science (Prato, 1997), biochemistry and biomedicine (Yang et al., 2009). Recently, carbon dots (CDs), emerging as a new class of fluorescent nanomaterials, have attracted considerable interest. Based on carbon skeleton structure, carbon dots usually exist in the size of less 10 nm, showing excellent properties such as ease of preparation (Jia et al., 2012; Liu et al., 2007), satisfactory fluorescent performance, low cytotoxicity and biocompatibility (Baker and Baker, 2010) owing to their specific nanometer dimension. Thus, CDs have been considered as a satisfactory candidate for biosensing (Wang et al., 2010), catalysis (Cao et al., 2011), and imaging (Wei et al., 2012; Zhao et al., 2011; Zhou et al., 2012). In the past few years, CDs were generally synthesized by two major methods. One way was known as top down, consisting of electrochemical oxidation (Zhao et al., 2008; Zhou et al., 2007), acidic oxidation (Dong et al., 2010), arc discharge (Xu et al., 2004) and laser ablation (Hu et al., 2009). Hydrothermal (Zhu et al., 2012), microwave (Salinas-Castillo et al., 2013) and ultrasonic (Zhuo et al., 2012), serving as another way
n
Corresponding author. Tel./fax: þ 86 23 68251225. E-mail addresses: [email protected], [email protected] (X. Yang). 1 Both authors contributed equally to this work.
http://dx.doi.org/10.1016/j.bios.2014.04.046 0956-5663/& 2014 Elsevier B.V. All rights reserved.
defined as bottom up, have been normally applied to synthesize CDs. Nevertheless, these methods exhibited several drawbacks such as tedious steps, toxic reagents, special equipments, strong acid (alkali) or high temperature and high costs, leading to their limitations for applications (Sahu et al., 2012). Therefore, exploring new methods for synthesizing CDs are still desired. As being well known, Fe3 þ served as an essential component of heme groups and one of abundant transition metals in human biological systems (Von Drygalski and Adamson, 2013). Currently, there exist various techniques for quantification of Fe3 þ including flame atomic absorption spectroscopy (Ajlec and Stupar, 1989), inductively coupled plasma mass spectrometry (Huang and Lin, 2001), atomic absorption/emission spectrophotometry (Wu et al., 2009), atomic fluorescence and UV spectrometry (Kok and Wild, 1960). However, most of these methods required sophisticated and expensive instrumentation and/or complicated sample preparation procedures and were time consuming (Lee et al., 2011; Wang et al., 2012; Xiang and Tong, 2006). In contrast, fluorescent sensors offered a simple, low-costly approach for assaying metal ions in biological and environmental samples. In this study, a simple, economical, and green method for preparing water-soluble CDs has been established with a quantum yield of 19.8%. Importantly, this strategy for producing CDs by one-step treatment of honey under low-temperature heating was reported for the first time (Fig. 1). In addition, both the precursor of CDs and the synthesis procedure are substantially environment friendly, leading to their biocompatibility and more extensive applications. Subsequently, we
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Fig. 1. Schematic illustration of synthesis of CDs and cells imaging (top) and coding (bottom).
applied the CDs for sensitive and selective detection of Fe3þ based on direct interactions between Fe3 þ and –COOH, –OH and –NH2 of CDs (Chinoporos, 1962; McIlwee et al., 2008; Moon et al., 2010), resulting in aggregations that facilitates the quenching of their fluorescence. Again, the practicability of this strategy has been validated by assaying Fe3þ in human blood samples. Meanwhile, the CDs were employed for cell imaging, coding and preparing fluorescent powder, suggesting their potential to broaden avenues for meaningful applications and commercial purpose.
2. Experimental 2.1. Chemicals and materials Honey was bought from Yonghui Supermarket (Chongqing, China). All the metal ions (Hg2 þ , Fe2 þ , Pb2 þ , Ag þ , Ca2 þ , Co2 þ , Mn2 þ , Sr2 þ , Zn2 þ , K þ , Na þ , Cu2 þ ) and trihydroxymethyl aminomethane (Tris) were obtained from Shanghai Sangon Biotechnonlogy Co., Ltd. (Shanghai, China). Disodium hydrogen phosphate (Na2HPO4) and sodium dihydrogen phosphate (NaH2PO4), 30% Hydrogen peroxide (30% H2O2), potassium dihydrogen phosphate (KH2PO4), citric acid and sodium citrate, glacial acetic acid (HAc), phosphoric acid (H3PO4), boric acid (H3BO3), sodium hydroxide NaOH), sodium chloride (NaCl), ammonium chloride (NH4Cl) were purchased from Dingguo Changsheng Biotechnology Co., Ltd. (Beijing, China). Ultrapure water, 18.25 MΩ cm, produced with an Aquapro AWL-0502-P ultrapure water system (Chongqing, China) was employed for all experiments.
2.3. Synthesis of CDs Basically, the green CDs were synthesized for the first time here. In brief, 2.0 g honey was added into 8 mL ultrapure water at the beginning. After stirring for mixing, this solution was introduced with 2 mL of 30% H2O2. Then the mixture was transferred into a 25 mL Teflon-lined stainless-steel autoclave. After heating at 100 1C for 2 h, the autoclave was cooled to room temperature, and this aqueous solution was adjusted to neutral with 2 M NaOH followed by filtered with 0.22 μm filter membrane to remove the larger product. Finally, the fluorescent carbon dots were collected by dialysis against deionized water though a dialysis membrane (1000MWCO) for 24 h. The powder of CDs was obtained by lyophilisation, and furhter dissolved in ultrapure water with the final concentration of 1 mg/mL. The CDs prepared here were stable for 3 months while stored in the dark at 4 1C. 2.4. Detection of Fe3 þ Firstly, 40 μL CDs (1 mg/mL), 40 μL Britton–Robinson (BR) buffer (50 mM, pH 6.0) and an appropriate volume of Fe3 þ working solution or sample solution were successively pipetted into a 1.5 mL vial. Subsequently, these solutions were diluted to 400 μL with Milli-Q purified water, and followed by vortex-mixed thoroughly. After reacting at 35 1C for 10 min, the mixtures were subjected to fluorescence measurements. Finally, interferences originated from other metal ions were investigated individually in the presence of CDs prepared here. 2.5. Preparation of blood samples
2.2. Instrumentation All fluorescence measurements were performed on a Hitachi F-7000 fluorescence spectrophotometer (Tokyo, Japan) with excitation slit set at 5 nm band pass and emission at 5 nm band pass in 1 cm 1 cm quartz cell. Meanwhile, UV/vis absorption spectra were recorded by a Shimadzu UV-2450 spectrophotometer (Tokyo, Japan). The high resolution transmission electron microscopy (HR-TEM) images were taken using a TECNAI G2 F20 microscope (FEI, America) at 200 kV. Elemental and functional groups analysis were obtained by an ESCALAB 250 X-ray photoelectron spectrometer and a Fourier transform infrared spectrometer (Tokyo, Japan), respectively. The quantum yields were obtained by using an Absolute PL quantum yield spectrometer C11347 (Hamamatsu, Japan). The powder of CDs obtained by lyophilisation in PiloFD84.3V (SIM, USA). Photographs of cells were taken with an Olympus fluorescence microscope 1 71 (Tokyo, Japan). A Fangzhong pHS-3C digital pH meter (Chengdu, China) was used to measure the pH values of the aqueous solutions and a vortex mixer QL-901 (Haimen, China) was used to blend the solution. The thermostatic water bath (DF-101s) was purchased from Gongyi Instrument Co., Ltd. (Henan, China).
Fresh human blood samples were originally collected from three healthy volunteers of Southwest University Hospital. In brief, 1 mL blood was initially dispensed in 5 mL of Red Blood Cell (RBC) lysis buffer, and incubated at room temperature for 15 min. Then, the lysed samples were centrifuged at 10,000 rpm for 10 min, and the supernatant were collected. Next, 0.2 mL 20% ascorbic acid was introduced into the above solutions to oxidize Fe2 þ to Fe3 þ for further analysis. Finally, the prepared samples were used for detections of Fe3 þ according to the general procedure without additional special treatment.
3. Results and discussion 3.1. Characterization of CDs To characterize this synthesized CDs, the maximum excitation and emission spectra of synthesized CDs were initially recorded as 338 nm and 420 nm (Fig. 2A) respectively, and the fluorescent properties of the CDs solution were subsequently investigated. The CDs aqueous solution emitted obvious blue fluorescence
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Fig. 2. (A) Fluorescence and UV–vis absorption spectra of CDs. Inset: photographs of CDs under daylight (I) and UV light (II). (B) Emission spectra of CDs for varying excitation wavelengths. (C)–(E) HR-TEM images of CDs.
Fig. 3. (A) FTIR of CDs; (B) XPS of CDs; (C) C 1s of XPS and (D) N 1s of XPS spectra of the CDs thus obtained.
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(photograph II) under UV light (365 nm) while appearing as completely colorless transparent under daylight (photograph I) in Fig. 2(A). Again, the UV–vis absorption spectrum showed a peak at 278 nm, owing to n–πn transition of CQO and π–πn transition of CQC (Fig. 2A). To address whether the synthesis of CDs showed excitation-dependent emission character or not, the emission peak shifted to longer wavelength and the fluorescence intensity decreased with the excitation wavelengths varying from 320 nm to 410 nm (Fig. 2B), suggesting the similar fluorescent properties. Besides, the as-synthesized CDs excited by 700 nm laser showed the maximum upconverted emission peak at 435 nm (Fig. S1), suggesting their infrared upconversion property, likely due to antiStokes photoluminescence (Ho et al., 2012; Mu et al., 2013). To further investigate the nanostructures of CDs, a high resolution transmission electron microscope (HR-TEM) was employed to directly observe the morphology and particle size distributions. As shown in Fig. 2(C) and (D), CDs obtained here existed as a majority population at the size of 2 nm and no aggregation emerged, depicting their satisfactory disparity. Meanwhile, the lattice spacing of 0.18 nm was accompanied with the [102] fact of graphitic carbon (Fig. 2E) (Wu et al., 2013; Zhu et al., 2012). Next, Fourier transform infrared spectroscopy (FTIR) and X-ray photoelectron spectroscopy (XPS) were applied to explore the surface groups, structure and components of these synthesized CDs in detail (Fig. 3). As revealed in Fig. 3(A), there existed O–H, N–H, C–H, CQN, CQO, CQC and C–O groups on the surface of CDs. Specifically, the absorption bands of O–H and N–H stretching vibrations appeared at 3380 cm 1 with C–H and CQN stretching vibrations at 2906 cm 1 and 2250 cm 1 respectively. Similarly, CQO and CQC stretching vibrations were at 1636 cm 1. Simultaneously, the peak at 1332 cm 1 was associated with C–H and N–H bending vibrations and the peak at 1071 cm 1 with C–O bending vibrations. To elucidate the components of the current
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CDs, XPS survey spectra were performed. As shown in Fig. 3(B), three major peaks of C, O and N obviously emerged in the spectrum, indicating that CDs prepared here were mainly composed of C (44.24%), O (45.95%) and N. To fairly describe the peak of N, the amplified peak is shown in Fig. 3(D). Furthermore, there were three peaks displayed at 284.5 eV, 286.6 eV and 287.9 eV (Fig. 3C), demonstrating that there existed C–C, C–O and CQN/ CQO on the surface of CDs, which further confirmed the previous FTIR data. Overall, the FTIR data showed satisfactory agreement with that of XPS, intensively suggesting that the CDs produced here were equipped with characteristic functional groups including –COOH, –OH and –NH2, thereby facilitating their excellent water solubility without any chemical modification.
3.2. Stability of CDs Again, to assess the stability of CDs for bearing circumstances, various experiments were performed. As revealed (Fig. S2A–S2C), the fluorescent intensities of CDs scarcely exhibited variation along with varying concentrations of NaCl, pH and time, demonstrating the CDs outstanding stability, tolerance for pH and photobleaching. Moreover,
Table 1 Recoveries of Fe3 þ in human serum samples detected by the proposed method (n ¼6). Sample
Added (nM)
Measured (nM)
Recovery (%)
RSD (%)
Human blood
0 20 40 60
102.30 121.50 141.20 163.20
– 96.00 97.25 101.50
1.19 0.98 1.12 1.08
Fig. 4. (A) Fluorescence spectra of CDs in the absence (black) and presence (red) of 100 μM Fe3 þ . Inset: photographs; (B) effect of different metal ions on the fluorescence intensity of CDs. Inset: visual observation; (C) fluorescence spectra of CDs in the presence of various concentrations of Fe3 þ ; (D) the relationship between F0 F and Fe3 þ form 800 μM to 5 nM. Inset: plot of the fluorescent intensity decrease (F0 F) versus the logarithm of concentrations of Fe3 þ added. (For interpretation of the references to color in this figure legend, the reader is referred to the web version of this article.)
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Fig. S2(D) indicated that as-synthesized CDs could remain stable in the majority of organic solvents, as different solvents exhibited little effect on this CDs. Taken together, a brand-new type of CDs was successfully synthesized by a developed simple and green method. Considering that this CDs have been functioned with –COOH, –OH and –NH2 on the surfaces, we asked whether they could potentially serve as a
fluorescent probe to detect Fe3 þ or not (Chinoporos, 1962; McIlwee et al., 2008; Moon et al., 2010). 3.3. Optimization of analytical conditions Towards that purpose, Fe3þ was introduced into the CDs as prepared. Fig. 4(A) shows that fluorescence intensity of the CDs at
Fig. 5. Hep-2 cells in the absence (A) and presence of 1 mg/mL CDs incubated for 30 min (B) or 1 h (C); Hela cells in the absence (D) and presence of 1 mg/mL CDs for 30 min (E) or 1 h (F); images of powders including NaCl (left vial) and CDs (right vial) under daylight (G) and UV light (H); images of absorbent cotton (I) and filter paper (J) unstained (left) and stained (right) by this CDs under UV light.
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420 nm decreases to about 50%, when 100 mM Fe3þ is added. Simultaneously, the CDs solution shifted to light yellow under visible light (photograph III) and no fluorescence emission was observed under UV light (photograph IV), indicating that the CDs may be applied for detecting Fe3 þ . To elucidate the interaction between Fe3 þ and CDs, HR-TEM was used to directly observe this interaction. The sizes of CDs increased dramatically in the presence of 100 mM Fe3 þ (Fig. S3), suggesting aggregations occurred. To determine Fe3þ effectively, conditions including pH, reaction time, temperature and buffers were optimized. Fig. S4(A) shows that the fluorescence intensity of CDs is constant in the absence of Fe3 þ while the pH values vary from 3 to 11, suggesting that CDs are stable in acid and alkaline environment. Nevertheless, the fluorescence intensity of CDs greatly decreased in the presence of 100 mM Fe3 þ , and ΔF reached maximum at pH 6.0. Likewise, effects of reaction time (1 min, 10 min, 20 min, 30 min, 40 min, 50 min) and reaction temperature in the range of 30–60 1C were investigated towards 100 mM Fe3þ (Fig. S4B and S4C), claiming that ΔF of the CDs were independent of both the reaction time and temperature. Finally, effect of various buffers such as Phosphate Buffer Solution (PBS), Citrate–HCl, Phosphate Buffer (PB), BR and Acetic Acid–Sodium Acetate Buffer (HAc–NaAc) were tested, and ΔF of the CDs only in BR buffer reached maximum rather than that in other buffers (Fig. S4D). Considering the speed, easy-operation and selectivity of this assay, pH 6.0, 35 1C, 10 min and BR buffer served as the optimal conditions during the following experiments.
non-toxicity. Again, benefited from their high fluorescence and low toxicity, CDs were further employed for cell imaging. Specifically, HEp-2 and Hela cells were incubated in the absence (Fig. 5A and D) and presence of 1 mg/mL CDs, and there exhibited weak (30 min incubation) and obvious fluorescence (1 h incubation) originated from CDs in both cell lines (Fig. 5B, C, E, and F), suggesting that the CDs described here may play a role for cell imaging. In addition, as shown in Fig. 5(H), the powder of CDs obtained by lyophilisation (right vial) showed striking fluorescence under UV light compared with NaCl powder (left vial), whereas no difference was observed between CDs and NaCl powder under daylight (Fig. 5G). Significantly, the fluorescent powder has been preserved for more than 3 months without marked changes, indicating that it may provide the possibility for matching the requirement of commercial scale. Finally, we also applied the CDs as fluorescent dye for applications. Interestingly, both absorbent cotton and filter paper with staining (right) implied dramatic fluorescence under UV light compared with that without staining (left) in Fig. 5(I) and (J). Moreover, this CDs, serving as fluorescent ink, were used for producing various paintings (Fig. S6A–S6C), and the words written by the chalk stained with this CDs showed clear fluorescence (Fig. S6D). Hence, these photographs strongly demonstrated that these fluorescent CDs could serve for imaging and staining.
3.4. Selectivity of analytical strategy
4. Conclusions
Next, the selectivity of this analytical strategy was evaluated by testing the response to other metal ions (500 mM for each) under optimum conditions for the case of 10 mM Fe3 þ (Fig. 4B). Stock solutions of other metal ions (Hg2 þ , Fe2 þ , Pb2 þ , Ag þ , Ca2 þ , Co2 þ , Mn2 þ , Sr2 þ , Zn2 þ , K þ , Na þ , and Cu2 þ ) were prepared. Fig. 4 (B) shows that the fluorescence probe responds selectively toward the other metal ions, indicating the excellent selectivity of the CDs for Fe3 þ detection. Furthermore, the calibration curve was constructed under optimum conditions. The fluorescent intensity decrease (F0 F) versus the logarithmic plot of Fe3 þ concentration displayed a linear range from 5.0 10 9 mol/L to 1.0 10 4 mol/L (Fig. 4C). As shown in Fig. 4(D), (F0 F) was in a linear relationship along with concentrations of Fe3 þ , and the linear regression equation is (F0 F)¼1295.80 147.02 C3Feþ with a correlation coefficient of 0.9937 (n ¼5). Additionally, the relative standard deviation (RSD) was 3.1% and 1.7% for five repeated measurements of 5.0 10 9 and 1.0 10 4 mol/L Fe3 þ respectively, demonstrating an excellent precision of this fluorescent probe. The detection limit of Fe3 þ was 1.7 10 9 mol/L at a signal-to-noise ratio of 3. Overall, the results showed a highly sensitive and widely linear range method.
In summary, we have creatively synthesized CDs based on honey via a simple and green method for the first time. Moreover, these CDs were employed as a novel sensor for the quantification of Fe3 þ , which depended on Fe3 þ coordinated with –COOH, –OH and –NH2 of CDs (Chinoporos, 1962; McIlwee et al., 2008; Moon et al., 2010), thus leading to aggregations that quench their fluorescence. Significantly, this new CDs described here were applied for cell imaging and fluorescent staining, demonstrating many broaden potential avenues in bioimaging, biosensing and other purposes.
3.5. Detection of Fe3 þ in real samples For testing the practicality of this developed approach, standard recovery experiments were performed in human blood samples (Table 1). As listed, the recoveries of all samples were 96.00%, 97.25%, 101.50% respectively, and there were little inference from substrates of the blood, indicating the proposed method may broaden ways for practical detections of Fe3 þ in real samples. 3.6. Fantastic applications Ultimately, two types of crops were introduced to explore the toxicity of the CDs as-synthesized here. The seeds of green soya beans (Fig. S5A), and water spinach (Fig. S5B), incubated with CDs regularly grew in pace with time-variation compared with that without CDs incubations, revealing the CDs satisfactory
Acknowledgments We gratefully acknowledge financial support by National Natural Science Foundation of China (31100981), Research Fund for the Doctoral Program of Higher Education of China (20110182120014), Natural Science Foundation Project of CQ CSTC (cstc2013jcyjA10117), Fundamental Research Funds for the Central Universities (XDJK201 3B038), and Program for Innovative Research Team in University of Chongqing (2013).
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