Economic and Ecofriendly Synthesis of Biocompatible Heteroatom

Research Article pubs.acs.org/journal/ascecg

Economic and Ecofriendly Synthesis of Biocompatible Heteroatom Doped Carbon Nanodots for Graphene Oxide Assay and Live Cell Imaging Santanu Patra,† Ekta Roy,† Rashmi Madhuri,*,† and Prashant K. Sharma‡ †

Department of Applied Chemistry and ‡Functional Nanomaterials Research Laboratory, Department of Applied Physics, Indian School of Mines, Dhanbad, Jharkhand 826 004, India S Supporting Information *

ABSTRACT: The present work reports an economic and eco-friendly strategy for fabrication of nitrogen doped fluorescent carbon nanodots (CNDs) by an electrochemical approach. Relative to previously reported approaches using harsh reaction conditions, the electrochemical approach requires less energy and reaction time for the preparation of highly stable CNDs. For the first time, four alkanolamines (ethanolamine, 3-amino-1-propanol, 4-amino-1-butanol, and 5-amino-1-pentanol) have been chosen for the preparation of CNDs, and it is found that with increase in chain length, the quantum yield (QY) value increases. The maximum QY of 51% was found for the CNDs derived from 5-amino-1pentanol. The as-prepared CNDs have very narrow size distribution and excellent water dispersibility. The CNDs were used for quantitative detection of a nanomaterial i.e. graphene oxide without any cross-reactivity. The label-free, fluorescence sensor was also applied for the detection of graphene oxide in environmental water samples and human blood and urine samples. To explore the multifacet of as-prepared CNDs, their cytotoxicity was also studied using MCF-7 cancer cells. It was found that even at very high concentration of CNDs (2000.0 mg L−1); more than 95% MCF-7 cells are alive. Furthermore, the internalization of CNDs to the MCF-7 cells was also studied using confocal fluorescence microscopy. KEYWORDS: Nitrogen-doped carbon nanodots, Electrochemical approach, Alkanolamines, Graphene oxide detection, Live-cell imaging, Cytocompatibility assay

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INTRODUCTION We are living in a world where in every minute of our life we are interacting with nanomaterials. Either they are in the form of medicines or used in electronic gadgets, scratch-free paints, sports equipment, cosmetics, food color additives, and/or water purifiers.1 According to a report released in 2012, more than 1000 consumer products having nanoparticles are available in the market and in demand too. 2 Our exposure with nanomaterials is significantly increasing day by day, yet there is very little knowledge we have about their toxicological properties and long-term effect on the health of any living creature.3 Because of their very nanoscale size, they are capable of entering the human body by inhalation, ingestion, skin penetration, or injections.4 While talking about the nanomaterials, the first, very fast growing name that came to light is graphene. It is a two-dimensional single sheet of carbon atoms with sp2 hybridization that has exceptional mechanical, optical, thermal, and electronic properties.5 Their application is very © XXXX American Chemical Society

common as an efficient adsorbent for the purification of water, a good sensory material, sorbent for solid phase microextraction, a super capacitor, catalyst, and a drug carrier for site specific delivery.6−10 It is reported in the literature that graphene production will reach to 573 tons in the year 2017.11 With such a high speed production rate, it can be assumed that they will very soon reach the environment through atmospheric emissions and waste streams from production and from research facilities. After their exposure to the environment, they can contaminate soil and surface ́ water and can easily enter into the eco-system. Beniteź Martinez et al. have summarized the toxicological effects of graphene oxide (GO), the most popular, easy-to-produce, and maximumly used form of graphene.12 According to them, GO Received: November 6, 2015 Revised: December 28, 2015

A

DOI: 10.1021/acssuschemeng.5b01446 ACS Sustainable Chem. Eng. XXXX, XXX, XXX−XXX

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ACS Sustainable Chemistry & Engineering

Table 1. Comparison of Different Parameters from the Earlier Reported Electrochemical Method for the Preparation of CNDs SN

preparation procedure

reaction time

application

ref

5.1−6.3

120 h long time

12.0

photocatalyst photocatalyst

27 28

4h

15−100 mA cm−2/6 V

15.9

cell imaging

29

8.5 h

80−200 mA cm−2

14.0

60 min 2h 2h 45 min

5V 5V 10 V 4V

11.9 10.04 27.1 51.0

biological label for stem cells photovoltaics detection of mercury ions detection of ferric ions detection of hemoglobin detection of graphene oxide and cell imaging

30 31 32 33 34 this work

electrochemical oxidation of MWCNTs

7−15 h

2 3

graphite rod as electrode graphite rod as electrode and ethanol/water as electrolyte electrochemical carbonizations of low- molecular weight alcohols electrolysis of the graphite rod graphene film as working electrode carbonization of sodium citrate carbonization of ethanol carbonization of glycine carbonization of alkanolamines

5 6 7 8 9 10

quantum yield (%)

1 V + 90 °C temperature 15−60 V 10−200 mA cm−2

1

4

required energy

26

temperature reflux method using various amino acids.21 Similarly, a number of approaches have been reported in the literature for the synthesis of heteroatom-doped CDs/CNDs namely hydrothermal,22 microwave irradiation,23 arc discharge,18 laser ablation,24 and oxidative acid treatment processes.25 The as-synthesized CNDs possess high QY, however, they require sophisticated and expensive energy consuming machinery and uncontrollable or harsh reaction condition and also suffer from a low degree of functionality, resulting in poor interaction with analytes,26 which limits their application to some extent. Recently electrochemical carbonization is becoming popular for synthesis of CNDs, due to their fast reaction in the absence of expensive chemicals, strong acids, and high temperature.26,27 The electrochemical synthesis of carbon dots involves a topdown, nonselective, and chemical cutting process using carbon precursors like graphite, carbon nanotubes in the form of modified electrodes (Table 1). The CDs synthesized using this approach of electrochemical synthesis result in mixed/large size CDs and require a further tedious separation process to obtain monodisperse CNDs. In addition to this, the resulting CDs have very poor QY, i.e. less than 20%, which limits their wide application in various fields. Therefore, a size-controlled, economic, speedy electrochemical technique is required for synthesis of high QY CDs. To resolve this, some researchers have synthesized CDs using the electrolyte of an electrochemical cell as a new carbon source. In the literature, three works have been reported using sodium citrate,32 ethanol,33 and glycine34 as electrolyte and precursor for electrochemical synthesis of carbon dots. However, a single work has been reported so far for synthesis of heteroatom-doped CNDs via an electrochemical route by Wang et al.34 They have synthesized nitrogen-doped CNDs using glycine as the raw material, and the CNDs were used for the detection of hemoglobin. Taking consideration of earlier reported literature works, for the first time, we have synthesized, here, a high quantum yield, nitrogen-doped CND (∼50%) by an electrochemical carbonization technique using four alkanolamines precursors namely ethanolamine (EA), 3-amino-1-propanol (APr), 4-amino-1butanol (AB), and 5-amino-1-pentanol (AP) (Figure S1). The reaction speed is so good that the CND preparation was completed within 45 min by applying a potential of 4.0 V. In addition, the required energy is much lower than that of earlier reported approaches (Table 1). Optimization of a suitable base (as a reaction medium) and the concentration, reaction time,

has the ability to penetrate through the plasma membrane, resulting in the change of cell morphology and oxidative stress with membrane damage in adenocarcinomic human cells.13 It is also reported that GO is highly toxic for human and animal cells, immune cells, and blood components, and according to Wang et al., GO can be accumulated in lungs, liver, and spleen of mice very easily for a long time.14 Therefore, their detection in biological fluids is very essential. To date, a number of approaches have been reported for the detection of various nanomaterials like carbon nanotubes and metal nanoparticles (gold nanoparticles, etc.).12 López-Lorente et al. reported a bare gold nanoparticle mediated surface-enhanced Raman spectroscopic method for the detection of carboxylated single-walled carbon nanotubes.15 The same group also reported an on-membrane Raman determination of singlewalled carbon nanotubes in another report.16 The same group also reported on the detection of gold nanoparticles in liver and river water samples.17 ́ ́ Recently, Benitez-Marti nez et al. have reported a graphene quantum dot sensor for detection of GO (limit of detection = 35.0 μg L−1). However, the sensor itself works on the fluorescence efficiency of quantum dots, which is a zerodimensional luminescent carbon-based nanomaterial consisting of very small graphene sheets.12 Therefore, a more efficient, ecofriendly, and economic sensor strategy required for sensing of trace level nanomaterials like GO in real samples is called for and an emerging topic of research. After the first synthesis and isolation of carbon dots or carbon nanodots (CNDs), in the year 2004, they became a very promising as well as most flexible nanomaterial in the field of sensing.18 In addition to sensing applications, they have acquired a lot interest in research fields like bioimaging, drug/gene delivery, and catalysis, within a very small time span of ten years, owing to their strong photoluminescence property (PL), biocompatibility, and high chemical and photostability.19 In general, the CNDs have a discrete, quasi-spherical shape and SP2 hybridization with a size below 10 nm.19 Their multifunctional behavior is due to the presence of several functional groups on their surface which can be modulated, fabricated, and modified on the basis of precursor compounds used during their synthesis. It was also found that the CNDs consisting of heteroatoms like nitrogen, phosphorus, and sulfur show more intense and stable fluorescence properties with high quantum yield (QY).20 Recently, in our laboratory, we have synthesized heteroatom doped CNDs of very high QY i.e. 38%, via high B


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ACS Sustainable Chemistry & Engineering Scheme 1. Synthesis and Multifunctional Behaviour of Nitrogen Doped Carbon Dots

purification. Ultrapure water (18.2 mV; Millipore Co., USA) was used throughout the experiment. UV−visible spectra of prepared CNDs were taken in a UV−visible spectrophotometer (Shimadzu, Japan; Model - UV 1800). The fluorescence spectra (FL) were recorded using a PerkinElmer LS55 fluorescence spectrometer. The size and morphology of prepared nanoparticles were studied using a transmission electron microscope (model Tecnai 30 G2 S-Twin electron microscope) operated at 300 kV accelerating voltages by placing a drop of as prepared sample suspension on the surface of a carbon-coated copper grid. EDAX spectra of the CNDs were recorded using field emission scanning electron microscope (FE-SEM), Zeiss, model Supra 55. The powder X-ray diffraction (XRD) study was carried by Bruker D8 Focus X-ray diffractometer instrument using a Cu target radiation source. Particle sizes were measured using a dynamic light scattering (DLS) instrument (Zetasizer nano-S90, Malvern Instruments Ltd., UK) with a He−Ne laser beam of wavelength of 633.8 nm. Preparation of Graphene Oxide. Graphene oxide (GO) was prepared by modified Hummer’s method reported earlier.7 In brief, graphite powder (1.0 g) was first oxidized with 20.0 mL mixture of HNO3 and H2SO4 (1:3, v/v) under reflux for 2 days, centrifuged, and washed with dry THF. The preoxidized graphite was further oxidized by a mixture of 96% H2SO4 (40.0 mL) and KMnO4 (5.0 g) and finally the GO was collected and dried under vacuum. Preparation of CNDs. The CDs were prepared via one-pot, simple electrochemical carbonization of four alkanolamines (EA, APr, AB, and AP), under basic and optimized analytical conditions. Prior to the synthesis, some parameters viz., selection of electrolyte, concentration of electrolyte, reaction time, and voltage were optimized and the optimized synthesis procedure is described here. For the synthesis of CNDs, two platinum sheets (30 mm × 30 mm × 0.1 mm) were used as the anode and cathode (Scheme 1). Both the electrodes were fixed at a distance of 50 mm, in an electrochemical cell (100.0 mL

and applied voltage was also done during the course of reaction. The as prepared CNDs were characterized by UV−vis, photoluminescence (PL) spectroscopy, X-ray photoelectron spectroscopy (XPS), powder X-ray diffraction (PXRD) analysis, transmission electron microscopy (TEM), and dynamic light scattering (DLS) analysis. CNDs show multiple applications in the field of cell imaging, patterning, and sensing. The confocal laser microscopic images were taken to explore the role of as prepared CNDs for the future-imaging agent in MCF-7 cancer cells. In addition the MTT assay was also employed to analyze the cytotoxicity of synthesized CNDs to live cells. In addition to these, exploiting the entirely different aspect of CNDs, we have used them for detection of a very popular nanomaterial i.e. graphene oxide (GO). The CNDs was successfully employed for trace level detection of GO over the concentration range of 9.99 ng L−1 to 1.19 μg L−1 with limit of detection = 3.33 ng L−1 at a signal-to-noise ratio of 3. It is found that the as-prepared CNDs could serve as a very effective fluorescent probe for label-free, sensitive, and selective detection of GO in environmental and human biological fluids like blood and urine samples, without any interfering effects. This stands for their future application in clinical trials.

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EXPERIMENTAL SECTION

Reagents and Instrumentation. Ethanolamine (EA), 3-amino-1propanol (APr), 4-amino-1-butanol (AB), and 5-amino-1-pentanol (AP) were purchased from Sigma-Aldrich (Germany). Sodium hydroxide, ammonium hydroxide, graphite powder, potassium permanganate (KMnO4), and other interferents were purchased from Spectrochem Pvt. Ltd. (India) and Loba Chemie Pvt. Ltd. (India). All chemicals were used as received without any further C


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Figure 1. (A) XRD and (B) FE-SEM image of prepared GO. (C) UV−vis spectra, (D) XRD pattern, (E) PL spectra, and (F) FT-IR spectra of synthesized CNDs.

Figure 2. Fluorescence spectra of (A) EA-CNDs, (B) APr-CNDs, (C) AB-CNDs, and (D) AP-CNDs at different excitation wavelengths (1, 300; 2, 313; 3, 325; 4, 338; 5, 350; 6, 363; 7, 375; 8, 388; 9, 400; 10, 413; 11, 425; 12, 438; and 13, 450 nm). (inset) Camera pictures of CNDs in the absence and presence of UV light. overnight at 65 °C for evaporation of the solvents. The obtained brownish powder was further suspended and dialyzed against pure water through a dialysis membrane (1000 Da MWCO) to get the pure CNDs (yield ∼ 51%). Based on the precursors used for synthesis of CNDs, they were named as EA-CNDs, APr-CNDs, AB-CNDs, and AP-CNDs, throughout the work. Determination of Fluorescence Quantum Yield. For the measurement of fluorescence QY, quinine sulfate (QS) dispersed in

capacity). As an electrolyte, a mixture of 47.0 mL of alkanolamine and 3.0 mL of NH4OH (3.0 M) was used. After assembly, a fixed voltage of 4.0 V was provided for carbonization of alcohols using direct current power source (Sigma DC regulated power supply, PS 3005). The preparation of CDs was confirmed by the change in color from colorless to light brown. After this, 50.0 mL of fresh ethanol was added to the solution to precipitate out the dissolved NH4OH and the whole mixture was centrifuged at 15 000 rpm. The supernatant was kept D


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Figure 3. (A) XPS survey spectra of AP-CNDs. The detailed (high-resolution) scans for (B) C 1s, (C) O 1s, and (D) N 1s spectra of AP-CNDs. were treated as blank control. After incubation for next 24 h, 20.0 μL of MTT was added to each well and the optical density was evaluated at 570 nm. Cell viability was calculated using the formula:

0.1 M H2SO4 was used as a standard.21 The QY of unknown CNDs was calculated according the following equation: QYX = QY stdIXA std nX 2 /IstdAX nstd 2

(1)

cell viability (%) = (A sample − A blank ) × 100%/(Acontrol − A blank )

Here I is the measured integrated emission intensity, n is the refractive index of the solvent, and A is the optical density. The subscript “std” is used for the standard sample with known QY, and “X” for the unknown samples. Detection of GO by Fluorescence Quenching Technique. For the detection of GO, 1.0 mL of CNDs solution (0.05 mg mL−1) was mixed with of various concentrations GO solution (10.0 μL). The change in fluorescence intensity after the addition of GO was measured. Under optimized condition (i.e., pH of the solution, binding time) the change in the fluorescence intensity of the solution with respect to the concentration of GO was calculated by Stern−Volmer equation. The linear relationship could be expressed by (I0/I ) − 1 = KSVC

(3) here Asample = absorbance of a well with cells, MTT solution, and CNDs; Ablank = absorbance of a well with medium and MTT solution, without cells; Acontrol = absorbance of a well with cells and MTT solution, without CNDs. The data was articulated as the percentages of viable cells compared to the survival of control (untreated cells as controls having 100% viability). Three parallel wells were run for each concentration and each experiment was repeated three times.

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RESULTS AND DISCUSSIONS Characterization of As-Prepared Graphene Oxide (GO). The prepared GO was characterized by XRD and FESEM analysis. As shown in the XRD pattern of GO (Figure 1A), a sharp and single peak at around ∼12.5° was observed, which belongs to (001) plane. This confirms the formation of pure phase GO.7 Furthermore, for the morphological study, FE-SEM images were taken which shows the clear sheet like morphology of synthesized GO (Figure 1B). In order to confirm the SEM observations, the size and thickness of synthesized GO sheets were also investigated using AFM technique. A representative AFM image along with Z-height profile is shown in Figure S2A. For AFM measurements, the suspensions of GO sheets were drop casted on a cleaned glass substrate. Very clear flakes of GO sheets are observed in the AFM image. The height profile (Figure S2B) shows the thickness of GO sheets as nearly 1.3 nm, which corresponds to the monolayer structure/thickness of GO sheets. This confirms the formation of single layered GO sheets. Characterization of Prepared CNDs. Spectroscopic analysis. Figure 1C shows the UV−vis spectra of prepared

(2)

where I0 is the initial fluorescence efficiency in the absence of GO, I is the fluorescence intensity after the addition of GO, KSV (L ng−1) is the Stern−Volmer quenching constant, and C (ng L −1 ) is the concentration of GO solution. The limit of detection (LOD) was calculated as three times the standard deviation for the blank measurement in the absence of template, divided by the slope of the calibration plot between [(I0/I) − 1] and concentration of GO. Cell Culture and Cytotoxicity Study of CNDs through MTT Assay. In vitro cytocompatibility study of CNDs was investigated using a standard methyl thiazol tetrazolium bromide (MTT) assay on MCF-7 (breast cancer cell line). For the cell culture, the MCF-7 cells were taken in the DMEM accompanied by 10% fetal bovine serum and 1% antibiotic solution. The cells were maintained at 37 °C in a humidified 5% CO2 atmosphere. For the MTT assay, first, MCF-7 cells were placed in 96-well plates at 37 °C in a humidified 5% CO2 atmosphere. After a 24 h interval, the cells were cured with various concentrations of CNDs (0−2000.0 mg L−1) and were cultured for another 24 h. Each concentration was added to three wells as parallel control and wells without cells as negative control. Wells without cell E


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(around 398.5 eV), and O 1s (around 531 eV) (Figure 3A). The deconvolutions of the C 1s spectra (Figure 3B) are fitted by two peaks, which are assigned to the presence of CC/C− C bond with a binding energy at 284.5 eV and C−N (289.4 eV). The O 1s peak can be deconvolved into three components (Figure 3C) attributed to the presence of −CO (536 eV), −C−O (532 eV), and −C−OH (526 eV) groups. The high resolution N 1s spectra (Figure 3D) reveal the presence of two nitrogen species of C−N (399.4 eV) and N−H (400 eV). Energy-dispersive spectrometry (EDX) and elemental analysis were performed to confirm the components of APCNDs. As shown in Figure S4, clear peaks for carbon and oxygen are observed, suggesting that the main element contained in the C-dots is carbon. Further elemental analysis was carried out, and as shown in Figure S4, the C-dots are composed mainly of C (58.32%), O (15.44%), N (13.14%), and H (13.10%). The zeta potential of the prepared CNDs were also evaluated and found as −22.3, −22.0, −21.6, and −21.0 for AP-CNDs, AB-CNDs, APr-CNDs, and EA-CNDs, respectively. The large negative charge on the alkanolamine derived CNDs may be explained on the basis of two electron rich functional groups i.e. −NH2 and −OH. However, the increase in the number of carbon chains does not the hamper the charge on the prepared CNDs, which implies that strong electrostatic repulsions will be present in the CNDs, resulting in their more stable dispersion and stability in solid state.35 The size distribution and morphology were characterized by TEM analysis (Figure 4). As shown in the figure, we can observe a spherical morphology of the CNDs and they are well separated from each other. AP-CNDs have a diameter ranging from 1 to 4 nm with average particle size of 1.5 nm. AB-CNDs have a higher average size ∼3.0 nm and APr-CNDs and EACNDs have average particle size 4.5 and 6.5 nm, respectively. The XRD spectrum is also in good agreement with the above results (Figure 1D). In the XRD spectra, one single peak at 18.1° (d = 6.85 nm) was obtained for AP-CNDs, indicating the amorphous nature of the prepared CNDs.36 In case of ABCNDs, APr-CNDs and EA-CNDs, the peak shifted to 18.2° (d = 6.76 nm), 19.1° (d = 6.45 nm), and 20.4° (d = 6.14 nm), respectively. This confirms the poor crystalline nature of carbon dots. Furthermore, the amorphous nature of CNDs was well supported by their selected area electron diffraction (SAED) pattern (Figure 4D, inset). The SAED pattern of AP-CNDs shows broad diffused rings with only one or two bright spots. Effects of Base, Applied Voltage, and Reaction Time on the Prepared CNDs. An alkaline environment is required for the formation of CNDs by the electrochemical carbonization process, for this first the effect of various base on the preparation of carbon dots was optimized. As shown in Figure 5A, NH 4 OH relative to NaOH and Na 3 PO 4 allowed preparation of CNDs with higher PL intensity. This may be due to the dual role of NH4OH i.e. as an N-doping precursor and a surface passivation reagent.28 After optimization of electrolyte for the reaction, their concentration was also optimized (Figure 5B). It was found that with increase in the concentration of NH4OH from 0.5 to 3.0 M, PL intensity get increases and decreases afterward. The decrease of PL intensity after 3.0 M may be due to the formation of more basic medium causing hindrance to the formation of negatively charged CNDs. Similarly, the other reaction parameters like applied voltage and reaction time were also optimized. The effect of applied potentials (1.0−6.0 V) on the PL intensity of CNDs is

Figure 4. TEM images of (A) EA-CNDs, (B) APr-CNDs, (C) ABCNDs, and (D) AP-CNDs. (inset) Their respective particle size distribution. (inset D) SAED image of AP-CNDs.

CNDs derived from all four different alkanolamine precursors. As depicted in the UV−vis spectra, a single peak was observed at ∼260 nm for all four CNDs. The peak may be attributed to the π−π* transition of the CC bonds and the n−π* transition of CO bonds present as a carbon core in the synthesized CNDs.21 To further explore the optical properties of CNDs, the PL spectra of all the CNDs under excitation wavelengths from 325 to 580 nm are measured and shown in the Figure 2A−D. With increase in the excitation wavelength the PL intensity first increased and then decreased with shift toward red indicating that both PL intensity as well as peak position is dependent on the excitation wavelength. It was also found that the excitation at 313 nm gives the maximum PL intensity in all the CNDs. Therefore, wavelength of 313 nm is optimized for the excitation and further PL study. The camera pictures of all prepared CNDs in the presence and absence of UV light is also shown in the inset of PL data (Figure 2, Inset). To explore the role of precursors on the fluorescence intensity of prepared CNDs, the all four CNDs were excited with 313 nm radiation and the corresponding PL spectra was recoded (Figure 1E). As shown in the figure, the PL intensity was observed in the order given below: EA-CNDs < APr-CNDs < AB-CNDs < AP-CNDs. Similarly, their QY values were also calculated using eq 1 with quinine sulfate as standard. The QY for AP-CNDs, AB-CNDs, APr-CNDs, and EA-CNDs were found to be 51%, 38%, 20%, and 7.7%, respectively. Based on the high quantum yield of AP-CNDs (i.e., derived from 5amino-1-pentanol), their role in various field has been exploited in this work. For the investigation of surface functional groups, FT-IR spectroscopy was carried out for all the four CNDs (Figure 1F and S3). It was found that all the CNDs exhibit some distinct absorption band at ∼3500 cm−1 (−O−H stretch), 3250 (N−H stretch), 2800 cm−1 (C−H stretch), 1650 cm−1 (CC stretch), 1396 cm−1 (C−C stretch), 1190 cm−1 (C−O stretch), and 1103 (C−N stretch), which confirms the successful incorporation of heteroatom (−N−) in the CNDs composition. The XPS study was also done to demonstrate the presence of functional groups in CND. The XPS survey spectra of CNDs show three typical peaks of C 1s (around 285 eV), N 1s F


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Figure 5. PL spectra for the optimization of suitable base (A) and concentration of base (B), applied voltage (C), and reaction time for the preparation of CNDs (D). Stability study of as-prepared CNDs against change in the (E) pH, (F) ionic strength, (G) UV radiation (365 nm), and (H) storage time at room temperature.

displayed in Figure 5C. It was observed that, with increase in the applied potential the QY value initially get increases, until 4.0 V, and afterward start to decrease. This may be due to the fast carbonization of precursor molecules, resulting in large and poor QY CNDs. Reaction time or electrolysis time is another important parameter for the preparation CNDs (Figure 5D). For this a 10.0−90.0 min interval was chosen with an optimal reaction time of 45 min. The PL intensity of CNDs increases with increase in the reaction time/electrolysis time up to 45 min, mainly because of the formation of larger amount CNDs.

On further increasing the reaction time, large sized and less dispersible CNDs were formed. Stability of the CNDs. For their potential future applications, the stability of the as-prepared CNDs must be explored. For this, the stability of the prepared CNDs was measured with respect to the change in their PL intensity under extreme pH, high ionic strengths, long time illumination, and storage conditions and is shown in Figure 5E−H. To study their stability against the pH, pH values were changed from 2 to 10 and it was found that the CNDs has a very good stability in G


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Figure 6. (A) Change in the PL intensity with increase in concentration of GO in the concentration range of 9.99 ng L−1 to 1.19 μg L−1. (B) Effects of various interferents on the detection of GO. (C) Proposed binding mechanism between GO and CNDs.

strong acid as well as strong alkaline conditions, as the PL intensity remains almost constant from pH 2 to 10 (Figure 5E). To measure their stability with increase in ionic strength, NaCl medium was taken as standard and their concentration were changes. It was observed that, the presence of highly concentrated NaCl solution (1.0 M) did not have any effect on the PL intensity, which suggests the high stability of prepared CNDs (Figure 5F). When the CNDs were placed under UV light illumination (365 nm) for 0.5 h, the change in PL intensity is about 1% (Figure 5G). However, while storage of CNDs was conducted at room temperature for three months, no obvious change in PL intensity was observed, which reveal the good photostability and storage ability of the prepared CNDs (Figure 5H). Quantitative Determination of GO. Prior to the detection of target analyte, the experimental conditions like binding time and solution pH were optimized (Figure S5 and S6). The GO detection was performed in a phosphate buffer solution (PBS), so, first their pH was optimized. As shown in Figure 6A, it has been observed that at basic pH (pH = 10), PLquenched efficiency reaches the highest value and afterward become constant. Similarly, the binding time between CNDs and GO was also optimized and found that 60 s binding time is sufficient to reach the maximum PL-quenched value. The low contact time value specifies that the quenching kinetic is fairly fast, which may be owing to the presence of both −OH and −NH2 groups on the surface of CNDs.26 Therefore, the PBS of pH 10.0 is used in the experiment with a binding time of 60 s. Under the optimized parameters, the AP-CNDs were applied for the detection of GO. For the analysis, a fixed concentration and volume of CNDs solution was taken in a glass vial, followed by addition of fixed volume but different concentration of GO. It has been found that with increase in the concentration of GO, the PL intensity get quenched linearly up to a certain point and thereafter become constant owing to the saturation in binding interaction between CNDs and GO. A good linear

Table 2. Detection of Graphene Oxide in Real Samples Using AP-CNDs samples sample 1 sample 2 sample 3

sample 1 sample 2 sample 3

sample 1 sample 2 sample 3

sample 1 sample 2 sample 3 a

added (ng L−1) 56.81 223.46 28.41 56.81 28.41 223.46 56.81 223.46 28.41 56.81 28.41 223.46 56.81 223.46 28.41 56.81 28.41 223.46 56.81 223.46 28.41 56.81 28.41 223.46

determined value mean ± SDa (ng L−1) Water Samples 56.72 ± 0.60 224.01 ± 2.24 28.82 ± 0.35 57.14 ± 0.63 28.42 ± 0.32 221.78 ± 2.55 Blood Serum Samples 56.82 ± 0.72 225.13 ± 2.64 28.15 ± 0.33 57.45 ± 0.63 28.45 ± 0.28 222.74 ± 2.25 Blood Plasma Samples 56.77 ± 0.69 221.91 ± 2.27 28.15 ± 0.28 57.00 ± 0.73 28.58 ± 0.38 223.48 ± 2.95 Urine Samples 56.81 ± 0.80 224.67 ± 2.39 28.85 ± 0.28 56.64 ± 0.67 28.22 ± 0.39 222.98 ± 2.72

Standard deviation of three replicate values. deviation.

b

recovery (%)

RSDb (%)

99.8 100.2 101.4 100.5 100.0 99.2

1.1 1.0 1.2 1.1 1.1 1.1

100.0 100.7 99.1 101.1 100.1 99.6

1.3 1.2 1.2 1.1 1.0 1.0

99.9 99.3 99.1 100.3 100.6 100.0

1.2 1.0 1.0 1.3 1.3 1.3

100.0 100.5 101.5 99.7 99.3 99.8

1.4 1.1 1.0 1.2 1.4 1.2

Relative standard

H


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Figure 7. Cytotoxicity assay of (A) EA-CNDs, (B) APr-CNDs, (C) AB-CNDs, and (D) AP-CNDs against MCF-7 cells with increasing concentrations from 0 to 2000 mg L−1.

herein we have taken multiwalled carbon nanotube (MWCNT), single walled carbon nanotube (SWCNT), gold nanoparticles (AuNP), and some metal ions Fe3+, Zn2+, Na+, K+, and Cd2+ as interfering substances. Figure 6B demonstrated the interference effect on the detection of GO using AP-CNDs as fluorescence sensor. As shown in the figure, no significant decrease in fluorescent intensity of CNDs was observed in the presence of any of the interfering substance, even at their high concentration. This may be attributed to the selective binding affinity of CNDs toward GO, which gives high selectivity to the proposed fluorescence sensor. Binding Mechanism between CNDs and GO. To study the binding between CNDs and GO, UV−vis spectra were taken for CNDs, GO, and CNDs-GO and represented in Figure S8. As from the spectra, it can be observed that CNDs and GO shows a single peak at 260.95 and 256.30 nm, respectively. However, after addition of GO to the CNDs solution, another peak appeared at 303.47 nm, which is redshifted to ∼40 nm from GO or CNDs. The appearance of single shifted peak in the spectra supports a bonding interaction between GO and CNDs. Based on the various studies, the schematic representation showing binding interaction between CNDs and GO is shown in the Figure 6C. The CNDs have mainly the −NH2 and −OH functional groups on their surface. However, the GO also possess the −OH moieties on their sheets, which may cause a good noncovalent majorly the hydrogen bonding interaction between them. In addition to these the core of CNDs were sp2 hybridized, which can also get participated in strong binding interaction with GO sheets. Therefore, the two strong interaction may be present in the solution, when GO were added to the CNDs solution, resulting in a sharp and fast quenching of the fluorescence. The strong binding interaction is also a strong cause for the better selectivity of sensor toward GO, even in the presence of interfering compounds.

Figure 8. Confocal fluorescence microscopic images of MCF-7 cell in the presence and absence of AP-CNDs, after 0 min (A and B) and 30 min incubation (C and D), respectively.

correlation (R2 = 0.99) was observed over the concentration range of 9.99 ng L−1 to 1.19 μg L−1, and the limit of detection is calculated to be 3.33 ng L−1 at a signal-to-noise ratio of 3 (Figure S7). The calibration equation was found as [(I0/I) − 1] = (0.002 02 ± 0.000 05) C (ng L−1) − (0.105 45 ± 0.028 71), n = 15, R2 = 0.99, with a Stern−Volmer constant of 0.002 02 L ng−1. Selectivity Study. Selectivity is one of the important parameter for any sensing device. To study the selectivity, I


Research Article

ACS Sustainable Chemistry & Engineering Real Sample Analysis. In order to validate the proposed method, the real water samples collected from different local area were analyzed. The water samples were collected from Damodar river, Dhanbad coal mine area, and Durgapur steel plant area. However, the human biological fluids were also analyzed to explore the effect of complex matrix on the detection of GO. The human blood serum, plasma, and urine samples were collected from local pathological laboratory. The real sample analysis was performed by standard addition method and the results were portrayed in the Table 2. As depicted in the table, approximately 100% recovery with 1−2% RSD was obtained in each sample. The results indicate that the proposed sensor is capable of analyzing the trace/ultratrace amounts of GO in real environmental water samples and human blood serum, plasma, and urine samples. Cytotoxicity Study. To further explore the important feature of the AP-CNDs in the field of cell imaging, their cytotoxicity study was performed with MCF-7 cells (Figure 7). In addition, to visualize the internalization of the CNDs to the cancer cells, their live cell imaging was also performed using confocal fluorescence microscopy (Figure 8). For cytotoxicity study, the MTT assay was performed with various concentrations (0−2000.0 mg L−1) of all the CNDs, prepared in this work. As shown in the figure, after incubation with 2000.0 mg L−1 of the CNDs for 24 h only ∼5−10% decrease in the cell viability was observed. This shows the low toxicity of CNDs, even in very large concentration was incubated with live cells. According to the literature, the prior studies of CDs/CNDs cytotoxicity have rarely included the concentrations beyond 1500.0−2000.0 mg L−1 (Table S1). In comparison to the earlier reported CDs/CNDs, the proposed CNDs show much better cytocompatibility toward live cells. This implies the future applicability of prepared CNDs as a cell-imaging probe. Furthermore, to examine the potential application of CNDs as a bioimaging probe, MCF-7 cells were cultured in the medium containing 0.05 mg mL−1 CNDs for 0.5 h at 37 °C, and then, the cells were observed under confocal microscope. The obtained bright-field image after incubation implies the viability of the cells (Figure 8). In the small time span of 30 min, it was observed that CNDs were very well interacted with the MCF-7 cells and a strong, bright green fluorescence was obtained, indicating the excellent cell membrane permeability of CNDs. According to the literature, the CDs synthesized by electrochemical process were mainly used in sensing; however, some of the researchers have applied them for cell imaging too, as shown in Table 1. From the study, it may be concluded that the CNDs derived from the green-synthesis approach are potentially safe and possess wide application in the field of in vitro and/or in vivo cell imaging.

without any interfering effect. In addition to these, the in vitro cytotoxicity assay against MCF-7 cells proves that as-prepared CNDs are not toxic to the live cells even at extremely high concentrations. To explore the in vivo uptake of CNDs by live cells, their confocal images were also recorded and found that proposed CNDs have good potential for live cell imaging also.

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ASSOCIATED CONTENT

* Supporting Information S

The Supporting Information is available free of charge on the ACS Publications website at DOI: 10.1021/acssuschemeng.5b01446. Chemical structure of precursors compounds used for synthesis of CNDs; tapping mode AFM image of GO and height profile; magnified (FT-IR spectra) of CNDs; EDAX spectra of CNDs; optimization of contact time and pH; calibration plot for GO detection; UV−visible study; table for comparison of cytotoxicity of CNDs (PDF)

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AUTHOR INFORMATION

Corresponding Author

*E-mail: [email protected]. Tel.: +91 9471191640 (R), +91 326 2235935 (O). Fax: +91 326 2296563 (R.M.). Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS The authors are thankful to DST, BRNS, and ISM for sponsoring the research projects to R.M. (ref no.: SB/FT/CS155/2012; FRS/43/2013-2014/AC; 34/14/21/2014-BRNS) and P.K.S. (ref no.: SR/FTP/PS-157/2011; FRS/34/20122013/APH; 34/14/21/2014-BRNS).

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REFERENCES

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CONCLUSIONS In summary, an electrochemical approach was applied for the preparation of water-dispersible and highly fluorescent Ndoped CNDs from four alkanolamines. The electrochemical approach requires a very low applied voltage (4.0 V) and shorter reaction time (45 min) than other previously reported methods. It was also found that with increase in chain length, QY of CNDs is also increasing and the maximum QY of 51% was found for AP-CNDs. Herein, we have demonstrated the label-free sensing of a very interesting and new target analyet i.e. GO, which is a very popular nanomaterial. The AP-CNDs have shown very good recovery and RSD, during quantitative estimation of GO in the water, human blood and urine samples, J


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