Synthesis of green fluorescent carbon quantum dots using waste polyolefins residue for Cu2+ ion sensing and live cell imaging

Accepted Manuscript Title: Synthesis of green fluorescent carbon quantum dots using waste polyolefins residue for Cu2+ ion sensing and live cell imaging Authors: Archana Kumari, Amit Kumar, Sumanta Kumar Sahu, Sanat Kumar PII: DOI: Reference:

S0925-4005(17)31282-0 http://dx.doi.org/doi:10.1016/j.snb.2017.07.075 SNB 22734

To appear in:

Sensors and Actuators B

Received date: Revised date: Accepted date:

24-3-2017 7-7-2017 12-7-2017

Please cite this article as: Archana Kumari, Amit Kumar, Sumanta Kumar Sahu, Sanat Kumar, Synthesis of green fluorescent carbon quantum dots using waste polyolefins residue for Cu2+ ion sensing and live cell imaging, Sensors and Actuators B: Chemicalhttp://dx.doi.org/10.1016/j.snb.2017.07.075 This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.

Synthesis of green fluorescent carbon quantum dots using waste polyolefins residue for Cu2+ ion sensing and live cell imaging Archana Kumaria,b*, Amit Kumarc, Sumanta Kumar Sahuc and Sanat Kumara,b* a

Academy of Scientific & Innovative Research (AcSIR), New Delhi, India.

b

CSIR- Indian Institute of Petroleum (CSIR-IIP) Dehradun-248005, India.

c

Department of Applied Chemistry, Indian Institute of Technology (ISM) Dhanbad -826004,

Jharkhand, India. *Corresponding author: [email protected] (Archana Kumari), Tel: +91 01352525794

Highlights 

A highly green fluorescent carbon dots was synthesized from waste plastic residue.

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The synthesized non toxic carbon dots were employed for the detection of the Cu2+ ions and live cell imaging.

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Limit of detection (LOD) of Cu2+ ions is 6.33 nM.

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The Cu2+ ions are also used for cell imaging of triple negative breast cancer cells (MDAMB 468 cells) and real water samples.

Abstract The residue obtained from the pyrolysis of waste polyolefins, has been used for preparation of highly green-visual fluorescent carbon quantum dots (CQDs) by a simple one step hydrothermal approach consisting of ultrasonic-assisted chemical oxidation. These CQDs were characterized by UV–Vis absorption spectroscopy, fluorescence spectroscopy, TEM, XRD, and FTIR. The CQDs possessed high stability in aqueous solution and exhibited strong fluorescence

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with quantum yield of 4.84%. The use of these CQDs as a fluorescent sensor for Cu2+ ions detection has been explored. The synthesized CQDs have excellent selectivity and sensitivity towards Cu2+ ions with a limit of detection (LOD) 6.33 nM and linear detection range of 1–8.0 μM. These CQDs have also shown their utility for analysis of real water samples and have the potential to use for triple negative breast cancer cells (MDA-MB 468 cells) imaging.

Keywords: Cancer cell imaging; CQDs; Cu2+ ions sensing; real water samples analysis; waste polyolefins residue.

1. Introduction Plastics are synthesized organic materials, having a high molecular mass and mostly containing carbon and hydrogen. The continuously increasing consumption of plastics have led to the generation of enormous amount of waste plastics. Due to their non biodegradable nature, the disposal of waste plastics in such a huge amount has become a serious problem for environment. To overcome this, utilization of plastic wastes as a cheap source of raw materials for value added products has become a predominant subject over all countries. Therefore, conversion of waste plastics to useful chemical feed stocks by chemical recycling are used as a solution to their growing environmental problem and as a viable alternative to fossil fuels and useful chemicals. Many researchers have worked on pyrolytic degradation of plastics taking virgin HDPE (High density polyethylene), LDPE (Low density polyethylene), PP(Polypropylene), PS (Polystyrene) and municipal plastics wastes as feed [1-3]. The use of virgin plastics predominantly results in high yield of liquid hydrocarbon and only about 1-2% of residue remains in pyrolyzer [4]. However, in the case of pyrolysis of used plastics (municipal

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waste plastics) a comparative larger amount of residue remains in pyrolyzer [5]. The municipal solid waste plastics consists predominantly of polyolefins (polyethylene and polypropylene to the extent of 60-70 %) [6] and hence most of the waste plastics conversion processes use segregated waste polyolefins as feedstock. In the case of pyrolysis of used polyolefinic waste plastics the amount of residue remaining in pyrolyzer could be of the order of 30-40 %. These residues have no use at all and as polyolefinic plastics are hydrocarbons. However since these waste plastics devoid of any hetero atoms (e.g. halogen, oxygen etc), their residues are also rich in carbon and can be used as a carbon source for synthesis of carbon quantum dots. Carbon dots or carbon quantum dots (CQDs) are generally defined as small carbon nanoparticles with various forms of surface passivation. Carbon quantum dots have low toxicity and high stable chemical properties which make them powerful candidates for new types of fluorescent probe. Tremendous efforts have been made on the synthesis of photoluminescent carbon nanoparticles (CQDs) using various precursors and methods, like laser ablation [7], arc discharge [8], acidic exfoliation and oxidation [9-11], hydrothermal/solvothermal treatment [1215], microwave irradiation [16] as they have unique properties and potential applications in bioimaging [17-19], drug delivery [20], metal ion sensing [13, 14, 21-25, 35-39] and photocatalysis [26-29]. Amongst the various precursors, the waste materials can provide advantages in terms of significantly reducing production cost and considerably saving resources. The use of CQDs as probe for copper ions, which plays a critical role in the areas of environmental, biological and chemical systems, has already been reported [13]. It is an essential trace element in various biological processes viz., embryonic development, mitochondrial respiration, regulation of hemoglobin levels as well as hepatocyte and neuronal functions. However, it may exhibit high toxicity and can cause damage to the central nervous system and

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disorders associated with neurodegenerative diseases. There are several methods which are used for detection of Cu2+ ions like atomic absorption spectroscopy (AAS) [30], inductively coupled plasma mass spectroscopy (ICP-MS) [31], ICP-AES (atomic emission spectroscopy) [32], and some electrochemical based methods [33]. Although the sensitivities of these developed methods for Cu2+ ions are generally high, most of these methods need either expensive or specialized instruments or sophisticated assay procedures, which greatly limit their wide applications. In the past decade, the organic dye/organic complex fluorophore-based probe as fluorescent probes [34] have been used to selectively detection of Cu2+ ions due to their relatively high sensitivity. However, the employed organic dyes/complexes are susceptible to oxidation, toxic and often have low photostability, which restrict their performance in practical applications. Therefore, there is still a need to develop simple and efficient Cu2+ ions sensor in biological and water environment. Some excellent works on CQDs as a probe for Cu2+ ions detection based on PL quenching have been reported. Liu et al. first developed polymer nanodots for Cu2+ ions sensing which exhibit a blue fluorescent emission [13]. They explored the feasibility of photoluminescent polymer nanodots for Cu2+ ions detection. The detection range is from 0-50 μM with a limit of detection (LOD) of 1 nM, and their response time to Cu2+ ions is 10 min. Wang et al. [37] synthesized blue-fluorescent graphene quantum dots via a hydrothermal reoxidation of graphene oxide to detect Cu2+ ions, the calibration curve of which displays a nonlinear region of 0- 15 μM with a limit of detection 0.226 μM. Liu et al. [38] and Dong et al. [35] synthesized BPEI-capped CQDs (BPEI-CQDs) and used as probe for selective and sensitive detection of Cu2+ ions. Liu et al. synthesized BPEI-capped CQDs by coating the as-prepared CQDs from bamboo leaves with BPEI and found LOD of 115 nM in the detection range of 0.333 to 66.6 μM whereas Dong et al synthesized BPEI-capped CQDs using citric acid and BPEI and

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found LOD of 6 nM in the detection range of 10-1100 nM. Hu et al. [36] synthesized CQDs from coal by acidic oxidation and used in the detection of Cu2+ ions. They found LOD of 2 nM in a detection range of 0- 50 μM. Wang et al. [39] synthesized yellow visible fluorescent CQDs from petroleum coke and found LOD of 29.5 nM in the linear detection range of 0.25 to 10 μM. In the present work, the pyrolytic residue (abbreviated in this paper as WP Residue) from a process converting segregated waste pololefins to diesel , has been utilized to prepare a new kind of green fluorescent CQDs , which shows a good stability, high selectivity, sensitivity and fast response for Cu2+ ions detection and cell imaging without any surface passivation. 2. Material and methods 2.1 Materials Waste polyolefin pyrolysis residue has been taken as carbon precursor. All other chemicals used were analytical-grade pure. 98.0 wt. % sulfuric acid, 65.0 wt. % nitric acid, 28.0 wt. % ammonia were used for the synthesis of CQDs. Aqueous solutions of Ni2+, Pb2+, Co2+ and Na+ were prepared from their nitrate salts, aqueous solutions of Mg2+, Ca2+, Hg2+, Sn2+ and K+ were prepared from their chloride salts and aqueous solution of Cu2+, Cd2+ and Al3+ were prepared from their sulphur salts. Nitrate, chloride and bromide salts of Cu2+ ions were also used. 3500 Da molecular weight cut off (MWCO) membranes was procured from Sigma Aldrich. The water used throughout the experiments was deionized (DI) water. 2.2 Synthesis of CQDs CQDs were obtained from residue left in pyrolyzer after the pyrolysis of waste polyolefin via the ultrasonic-assisted chemical oxidation approach [37]. 2 g residue was added into a

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mixture of concentrated H2SO4 (45 mL) and HNO3 (15 mL) and the solution was sonicated at 700 W in a flask for 2 h. The suspension was then transferred to a Teflon-lined autoclave (100 mL) and heated at 120 oC for 12 h. After the reaction, the mixture was cooled to room temperature (RT), then diluted ten times with DI water and adjusted to neutral with ammonia. The neutralized mixture was filtered with 0.22 μM membrane and dialyzed in a dialysis bag (MWCO 3500 Da) for 72 h to remove the remaining salts and tiny fragments to obtain a CQDs solution and was stored below 4 °C for further uses. The CQDs solution exhibited a green emission under UV light at 365 nm, shown in scheme-1. In contrast to many reported methods [35, 43-45] no surface passivation agents and passivation steps have been incorporated in this synthesis route. 2.3 Fluorescence detection of Cu2+ ions by CQDs The CQDs have been applied for selective detection of Cu2+ ions at room temperature. Different kinds of metal ions (Al3+, Ca2+, Mg2+, Ni2+, Co2+, Pb2+, Cu2+, Sn2+, Cd2+, Hg2+, Na+ and K+) solutions have been used. In a typical experiment, aqueous metal salt solution (10 µM, 2 mL) was mixed with 2 mL of CQDs solution (1 µg/mL). The aqueous mixture of metal ion and CQDs was left for 10 min at room temperature and then the fluorescence spectra of the mixture were recorded. 2.4 Preparation of real water samples To demonstrate t the feasibility of CQDs for sensing of Cu2+ ions in real water samples, , two kinds of water sources weree used, i.e. bottled mineral water purchased from local market and running tap water from our laboratory. Both mineral water and tap water were spiked

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separately with different concentrations of Cu2+ ions (10 nM, 20 nM & 30 nM) and the fluorescence spectra of the resulting solution was then recorded. 2.5 pH dependent fluorescence spectra and ion strength stability of CQDs The effect of pH (2-11) on photoluminescence (PL) spectra of CQDs was observed using HCl (2 M) and NaOH (2 M) solution. Aqueous solution of NaCl with different concentration (0.5, 1.0, 1.5 & 2 M) was used to know the ion strength stability of CQDs. 2.6 Cell toxicity The MDA-MB 468 cells were harvested as well as seeded into 96-well plate (90 μL.well−1) for overnight. Then the cells were left for 24 h to grow, followed by addition of MTT solution towards each well. After that cells were incubated with different concentration of CQDs (25, 50, 100, and 200 mg mL-1) for 4 h, the excess culture medium was discarded, and 150 μL of extraction buffer was added. The obtained mixture was left in dark at 37 °C for 15 min, and its optical density (OD) was measured at 500- 520 nm by using a micro plate reader made by Biotek. The samples were analyzed three times. The cell viability was calculated by using the following equation (1): Cell viability (%) = (ODtreated/ODcontrol) X 100%

(1)

Where ODcontrol is optical density measured in the absence of CQDs, and ODtreated is optical density measured in the presence of the CQDs. 2.7 Cell imaging studies

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MDA-MB 468 cells ware cultured in Dulbecco’s Modified Eagle’s Medium (DMEM) containing 10% fetal bovine serum (FBS). MDA-MB 468 cells were seeded in a 35 mm plate and cell lines were maintained at 37 °C for 24 h in a humidified incubator containing 5 % CO2. After the removal of culture medium, the cells were incubated with fresh culture medium containing 100 μL of aqueous solution of CQDs (1 mg mL-1) for 8 h. After that, the cells were washed with phosphate-buffered saline (PBS) by three times and cell imaging was carried out on a fluorescence microscopy. 2.8 Characterization The functional groups of synthesized nanoparticles were investigated by FT-IR (Thermo Nicolet Nexus 8700 FTIR spectrometer). The XRD patterns of CQDs were recorded on Phillips PW 1710 X-ray diffractometer (XRD) with Cu-Kα radiation. The morphological structure of the nanoparticles was determined by TEM. The transmission electron microscopic (TEM) image, EDX and elemental mapping were obtained by the JEOL JEM-2100UHR microscope with an accelerating voltage of 200 kV. The hydrodynamic size of the nanoparticles was measured by dynamic light scattering (DLS) techniques, using a Malvern Instruments, UK. The UV–Vis absorption spectra were measured at room temperature by Shimadzu UV-2600 spectrophotometer. Excitation dependent emission spectra and PL excitation (PLE) spectra were measured using LS55 Perkin Elmer Fluorescence Spectrophotometer. The life time decay was measured on a Fluoro Max-4 fluorometer (Horiba Jobin Yvon Inc, France). An excitation wavelength of 490 nm was used and emission was collected at 540 nm. The cell imaging was carried out by fluorescence microscope (Hitachi 7400) for the detection of fluorescence intensity in MDA-MB 468 cells (excitation wave length of 330-550nm was used).

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2.9 Quantum Yield calculation The quantum yield of the CQDs in aqueous solution was measured using quinine sulfate as the reference (54%) and 490 nm as the excitation wavelength and calculated using following equation (2) [25] and found to be 4.84% (Fig. S-1).

Q Q I /I A /A  2 / 2 CQDs R CQDS R R CQDs CQDs R

(2)

Where, “Q” represents the quantum yield, “I” represents the fluorescent intensity, “A” refers the absorbance measured at exited wavelength, and “Ƞ” represents the refractive index of the solvent used. The subscript “R and CQDs” refers the reference sample of known quantum yield and carbon quantum dots respectively.

3. Results And discussions 3.1 Optical properties of CQDs The optical properties of CQDs were explored by using UV- visible spectroscopy and fluorescence spectrometer. The UV–Vis spectrum of CQDs is shown in Fig. 1(a) and corresponding PL spectrum of CQDs is shown in inset of Fig. 1(a). The photographs of the CQDs taken under day light and UV light (365 nm) are shown in inset of Fig. 1(a). In the UV– Vis spectrum of CQDs show a broad and weak absorption peak around 280 nm, which may be due to the π– π* electronic transition of C–O groups of the CQDs. Excitation dependent fluorescent spectra of synthesized CQDs are shown in Fig. 1(b). The maximum emission spectrum at 540 nm was observed when the excitation wavelength is 490 nm. At this particular wavelength (490 nm) maximum numbers of particles may be excited.

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It is observed that PL spectra correspondingly red-shifted from 420 to 550 nm and the PL intensity decreases with increased in excitation wavelength 420 to 530 nm. The PL spectra shifting of CQDs could be associated with the degree of oxidation and oxygen containing groups found on the surface of the CQDs. The UV-vis and PL spectra of synthesized CQDs at different temperature (100 °C, 120 °C and 140 °C) are given in figures 2(a) and (b) respectively. The maximum absorption and emission spectra were observed at 120 °C and further increase in temperature, absorption and emission spectra are decreased. The fluorescence stability of the synthesized CQDs is brownish in colour and remains stable for months are shown in Fig S-3. 3.2 Fluorescence study of CQDs at different pH and ion strength stability The influence of pH on the synthesized CQDs was studied in different buffer solution (pH 2- pH 11) are shown in Fig.S-4. The fluorescence intensity of the CDs was increased in presence of acidic medium (pH 2.0- pH 5), remained almost constant at (pH 6.0- pH 9.0) and then decreased in basic (pH 11) medium. This behavior may be due to some functional groups found on the surface of the CQDs (like C=O, -OH). Increase in fluorescence intensity at lower pH may be due to the behavior of C=O group as positive charge carrier (

) in lower pH

range, presence of more positive charge on the CQDs leading to a higher net surface charge that causes electrostatic repulsion and thus increase in PL intensity. It presents a good relationship in the acidic & basic range, which indicates that CQDs can have potential application as a pH sensor. The effect of the salt analysis on the CQDs was evaluated by using NaCl buffer solutions (0 to 2.0 M), which are shown in Fig. S-5. It was observed that on addition of Nacl buffer solution, there was no change in the PL intensities of CQDs, suggesting that these CQDs can be used in salt buffers solutions. The stability of the CQDs in salt solutions makes them suitable for application in sophisticated and harsh conditions. 10

3.3 Particle size and morphology analysis The actual particles size and shape of CQDs was obtained by TEM images. The results show that the CQDs are spherical shape and uniform in size, as shown in Fig. 2(a). The average diameters are 2.5 nm and the narrow size distribution ranging from 1.5-3.5 nm are shown in Fig. 2(b). The hydrodynamic diameters of CQDs are determined by DLS analysis is shown in Fig. 2(d). The obtained results show that the CQDs are about 70 nm in size, which may be due to the agglomeration of the particles. The elemental composition of CQDs was estimated by EDX analysis and is shown in Fig. 2(e), the weight % of C and O have been found to be 88.79 and 11.21 respectively. Fig. 2(e) also shows the elemental mapping of the CQDs, which revealed the presence of C and O in the synthesized CQDs. The high-resolution TEM (HRTEM) images show shows that the CQDs have lattice spacing with inter-fringes distance 0.21 nm, which is consistent with that of (100) diffraction planes of graphitic carbon as shown [inset of Fig. 2(a)] [36, 40, 41]. The results suggest that the CQDs are mainly composed of nanocrystalline cores of sp2 graphitic carbons [42]. The amorphous nature of the CQDs is also confirmed by SAED pattern as shown in Fig. 2(c). 3.4 XRD pattern of CQDs The degree of crystalline, phase purity and physical properties of the CQDs have been determined by the powder XRD. The XRD patterns of WP residue and CQDs are shown in Fig. 3a. The XRD pattern WP-residue show some sharp and intense peaks, however after formation of CQDs these peaks have disappeared and a new broad diffraction peak around 2θ = 28° was observed, which corresponds to (002) plane with interlayer spacing of 0.318 nm. This indicates

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that the CQDs are amorphous in nature, which is probably due to the presence of closely packed carbon atoms with surface functionalities [43, 44]. 3.5 FTIR Spectra of WP residue and CQDs The functional groups existing in the WP residue and synthesized CQDs have been characterized by FTIR spectra and are shown in Fig. 3(b). The FTIR spectrum of WP residue shows intense absorption peaks at 2915, 2850, 1458 and 811 cm-1, indicating the presence of aliphatic hydrocarbons in WP residue .The peak at 3430 cm-1 is due to presence of O-H group of water molecule absorbed by the WP residue. The FTIR spectrum of the CQD shows that four new peaks have appeared around 3200 cm–1, 1615, 1126 and 640 cm–1. The broad and intense peak at 3200 cm–1 can be attributed to O-H group and it indicates that the existence of hydrogen bonds as this has also shifted the absorption peak to lower wavelength. The other three peaks around 1615, 1126 and 640 cm–1 confirms the existence of carbonyl group (C=O stretching vibration), C−O bond (C-O stretching vibration) and O-C-O group (O–C–O bending vibration). The absorption peaks of C–H at 2924, 2850 cm-1 observed in WP residue have greatly decreased in CQD, which indicates thermo oxidative degradation of the large molecules of WP residue. 3.6 Analytical Performance of CQDs for Cu2+ ions sensing When Cu2+ ions are added into the CQDs, the fluorescence intensity considerably decreases. Fig. S-6 shows the time-dependent fluorescence quenching spectra of the CQDs in the presence of Cu2+ ions (10 μM). The results indicated that the fluorescence intensity decreases with increase in time from (2 to 15 min). The maximum fluorescent quenching of CQDs was observed at 10 min, thus 10 min was chosen as the detection time in the following experiments. Different concentrations of Cu2+ ions are added to the CQDs and the fluorescence spectra have been 12

measured at an excitation wavelength 490 nm, which are shown in Fig. 4(a). Various salts solution of Cu2+ ions were used to confirm that the quenching is caused by Cu2+ ions and not due to the associated anions, which are shown in Fig. S-7. The sensing effect of CQDs towards different anions, organic molecules and biomolecules are shown in Fig. S-8 and S-9. The selectivity of the Cu2+ ions have been confirmed by the addition of salt solutions of different metal ions (Cu2+, Al3+, Ca2+, Mg2+, Ni2+, Co2+, Pb2+, Cd2+, Hg2+, Sn2+, Na+ and K+) into the CQDs. A histogram has been plotted between (F/F0) and various metal ions in solutions of constant molar concentration (10 μM), and is shown in Fig. 4(b) where, F indicates the fluorescent intensity of CQDs in the presence of different metal ions and F0 indicates the fluorescence intensity of CQDs in the absence of metal ions. The least PL intensity ratio was observed in the presence of Cu2+ ions as compared to the other metal ions. Photographs taken under UV light after addition of different metal ions into the CQDs are shown in inset of Fig. 4(b). The photographs clearly shows the greenish fluorescent colour of the CQDs have been effectively diminished by the addition of the Cu2+ ions, which indicates that the CQDs works as a probe for the detection of Cu2+ ions. The fluorescence quenching spectra of Cu2+ ions have been analyzed by the Stern–Volmer equation (3): F0/F=KSV [Q] + 1,

(3)

Where F0 and F are the PL intensity of CQDs in the absence and presence of the Cu2+ ions, respectively, [Q] is the concentration of Cu2+ ions (10 μM) and Ksv is the Stern-Volmer quenching constant. This equation represents the relationship between the relative fluorescence intensity (F/F0) and Cu2+ ions concentration. The fluorescence intensity of the CQDs versus Cu2+ ions concentration

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does not show linearity for all concentrations as shown in Fig. 4(c). Experimental data fit in Stern-Volmer equation only in the range of 1-8 μM [inset of Fig. 4(c)]. The calibration curve shows a good linear correlation (R2=0.998) over the concentration range of 1-8 μM. Under this optimum condition, the limit of detection (LOD) was calculated to be 6.33 nM based on 3σ/K (where σ is the standard deviation of the blank signal of CQDs and k is the slope of the calibration curve) and the calculated LOD is much lower than the other previously reported value [36-38]. The comparison of the detection limits for Cu2+ ions based on different analytical methods are shown in Table S1. It is clear that the LOD of our assay protocol is lower than that of the other reported methods. 3.7 Possible mechanism of the fluorescence response of CQDs to Cu2+ ions CQDs possess a variety of functional groups and optical properties, which could bind targets through different mechanism such as, inner filter effect [35], aggregation induced emission [45] quenching or electron transfer [46, 47]. In this work, the possible mechanism was investigated by fluorescence lifetime, Zeta potential and UV spectra. The fluorescence lifetimes decay of the CQDs in the absence and presence of Cu2+ ions were measured by time-correlated single photon counting, with excitation and emission wavelengths of 490 and 540 nm, respectively as shown in Fig. 4(d). The fluorescence lifetime of the CQDs (the black line) is 5.6 ns and after the addition of Cu2+ ions (the red line), the lifetime of the CQDs decreased to 4.6 ns suggesting the occurrence of a dynamic quenching [48]. Fig. 2(c) shows the UV–Vis spectra of CQDs in presence and absence of Cu2+ ions. After addition of Cu2+ ions the UV–Visible absorption peak has undergone a blue shift from 280 nm to 256 nm. This may be due to the chelation of Cu2+ ions through carbonyl groups present on the surfaces of the CQDs. Moreover, Cu2+ ions have led to the decrease in zeta potential from 4.92 mV to -7.94 14

mV, as shown in Fig. 2(d), which can also be attributed to the chelation of Cu2+ ions by CQDs via oxygen functional group present on the surface [49, 50]. 3.8 Visual detection of Cu2+ ions Different concentrations of Cu2+ ions (2 µM, 4 µM, 6 µM, 8 µM, and 10 µM) were prepared from the stock solution (10 µM) and added into the CQDs. After five minutes, photographs of these solutions were taken under UV light. Fig 5(a) clearly shows that the color of the solution changed from dark green to light green when concentration of Cu2+ ions was increased. This indicated that the synthesized CQDs could be used for visual detection of Cu2+ ions also. 3.9 Analytical performance of CQDs for Cu2+ ions sensing in real water Application of CQDs for Cu2+ ions sensing in real water samples [mineral water and tap water] with different concentration of Cu2+ ions (10 nM, 20 nM, and 30 nM) are shown in Fig. 5(b) & (c). It can be clearly seen that the fluorescence intensity gradually decreases with increase in concentration of Cu2+ ions both in mineral water and tap water. Good fluorescence quenching was observed inspite of the interference from numerous minerals, which possibly existed in different water sources. These results show that CQD-based probe can be applied for an accurate analysis of Cu2+ ions a wide range of medium including mineral water and tap water. 3.10 Cytotoxicity assay Considering the utility of the CQDs as fluorescent probes for cell imaging, it is fascinating to explore their toxicity towards MDA-MB 468. Therefore, the cytotoxicity of the CQDs is further measured by the MTT method. The MDA-MB 468 cell viability is 99% at a CQDs concentration of 25 μg mL-1 and the cell viability decreased from 97% to 74% when the 15

CQDs concentration was increased from 50 to 200 mg mL-1 are shown in Fig. S-10. However there is very less cytotoxicity up to 200 mg mL-1 of CQDs, which indicates the low cytotoxicity and good biocompatibility of the CQDs. These results indicate that the CQDs are successfully used as probes for cells imaging with very less cytotoxicity. 3.11 MDA-MB 468 cell imaging by synthesized CQDs CQDs were used as probe for confocal fluorescence imaging of MDA-MB 468 cells. Cells were incubated for 4 hr. The cell became brightly illuminated with multicolor images exhibited blue, green and red emissions under 330−385 nm (ultraviolet), 450−480 nm (blue), and 510−550 nm (green) excitation wavelengths, respectively are shown in Fig. 6(a-c). The laser confocal microscopic images also suggested that, CQDs show good photostability and low photobleaching, as the fluorescence intensity of labeled MDA-MB 468 cells remains stable for 1 h (after constant excitation). Therefore, the CQDs acted as potential candidates for specific MDA-MB 468 breast cancer cell labeling and multicolor imaging. Conclusion In this work, simple and effective green fluorescent CQDs with tuned size have been synthesized for Cu2+ ions sensing and cell imaging with improved sensitivity. CQDs were prepared from the residue left in pyrolyzer after the pyrolysis of waste polyolefins, by an ultrasonic-assisted chemical oxidation method without any subsequent chemical modification. The CQDs have exhibited that these can be used as a sensing probe with a rapid response as compared to the already reported CQDs. Moreover these are highly selective and show no interference from other metal ions. The fluorescence intensity of the CQDs versus the concentration of Cu2+ ions has a good linearity in the range of 1-8 μM/L, and the limit of

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detection is 6.33 nM. The CQDs have been used as promising multicolor (blue, red and green) fluorescent probes for MDA-MB 468 breast cancer cell imaging. The CQDs prepared from WPresidue (as a fluorescent probes) also show good cell-permeability and low cytotoxicity, thus also can effectively be used for the fluorescence imaging of MDA-MB 468 cells. Acknowledgement: The authors are thankful to Director, CSIR-Indian Institute of Petroleum for permission to publish this work. Council of Scientific and Industrial Research (CSIR), New Delhi is acknowledged for financial support for this work. Indian Institute of Technology (ISM), Dhanbad is gratefully acknowledged for fluorescence study. Supporting Information Materials used; Quantum yield calculation; Photoluminescence spectra of synthesized CQDs after 1 day and 1 month; The influence of pH values on the PL intensity of the CDs; Effect of ionic strengths on the fluorescence intensity of CQDs (ionic strengths were controlled by various concentrations of NaCl solution); Fluorescence responses of CQDs in the presence of different anions (10 µM); Fluorescence responses of CQDs in the presence of different organic species and biomolecules (10 µM); MTT based cell viability values (%) of CQDs up to 200 mg/mL in MDA-MB 468 cell after 4 h incubation with different concentrations of CQDs at room temperature; Comparison of linear range and limit of detection (LOD) for detection of Cu2+ ions. References [1] M. A. Uddin, Y. Sakata, A. Muto, K. Koizumi, M. N. Zaki, K. Murata, thermal and catalytic degradation of municipal waste plastics into fuel oil. Polymer Recycling 2 (1996) 309–315.

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Biography Archana Kumari received her B. Sc. (Chemistry) from Maghadh University, Bodh Gaya (India) in 2008 and M. Sc. (Chemistry) from Patna University, India in 2010. Currently he is pursuing Ph. D in chemistry at CSIR, Indian Institute of Petroleum, Dehradun, under the supervision of Dr. Sanat Kumar. Her research interests focused on the thermal and catalytic degradation of waste plastics and synthesis of carbon material from waste plastics residue left after pyrolysis. Amit Kumar received his B. Sc. (Chemistry) from Maghadh University, Bodh Gaya (India) in 2007 and M. Sc. (Chemistry) from Patna University, India in 2010. Currently he is pursuing Ph. D in chemistry at Department of Applied Chemistry, Indian Institute of Technology (ISM) Dhanbad, under the supervision of Dr. Sumanta Kumar Sahu. His research interests focused on the synthesis of carbon dots based sensors and bio imaging. Dr. Sumanta K. Sahu received his Ph.D in nano-medicine from Indian Institute of Technology, Kharagpur in 2011. He is currently the head of the research group „Functional Nanomaterials for biomedical applications‟ at the Department of Applied Chemistry, Indian Institute of Technology (ISM), Dhanbad since 2012. He has co-authored over 40 scientific research papers. His research interests include nanomaterials, functional porous materials, and their applications in drug delivery, catalysis, sensing, and separation. Dr. Sanat Kumar holds a Ph.D in chemistry. He is currently the head of the Crude & Wax Rheology area, CSIR-Indian Institute of Petroleum (IIP). His research interests include utilization of waste plastics for various value added products, rheological study of waxy petroleum product, compatibility of various crude oil and chemistry and formulation of waxes.

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H2SO4, HNO3 0

120 C WP residue

CQDs CQDs in day in UV light light

After addition of 2+ Cu ions

Scheme 1: Schematic illustration for the synthesis of CQDs from WP residue.

Figure 1: (a) UV-vis absorption spectrum (in inset PL spectrum), and photographs of the CDs under daylight and UV (365 nm) light, (b) excitation dependent PL spectra of CQDs (c) UV-vis

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spectra of CQDs in presence of Cu2+ ions (d) Zeta potential of CQDs are in absence and presence of Cu2+ ions

Figure 2: (a) TEM image ( in inset HRTEM image) (b) The particles size distributions (c) SAED pattern (d) DLS measurement and (e) TEM-EDX [in inset elemental mapping of carbon(left) and oxygen(right)] of CQDs.

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Figure 3: (a) XRD pattern and (b) FTIR spectra of the WP residue and CQDs.

Figure 4: (a) PL intensity suppression of CQDs in the presence of different concentrations of Cu2+ ions, (b) corresponding bar diagram of different metal ions and inset corresponding photographs taken under UV light, (c) The relative fluorescence intensity of CQDs to the 27

concentration of Cu2+ ions [Inset: The linear fitting to the relationship of F/F0 versus Cu2+ ions concentration from 1-8 μM], and (d) Fluorescence decay curves of CQDs by TCSPC in the absence and presence of Cu2+ ions with excitation of 490 nm.

Figure 5: Fluorescence images of (a) Cu2+ ions [2 µM, 4 µM, 6 µM, 8 µM and 10 µM] under UV lamp (365 nm). The fluorometric detection of Cu2+ ions in real water samples at different concentrations of various water sources [(b) mineral water and (c) tap water] by CQDs.

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Figure 6: Confocal fluorescence microscopic images of MDA-MB 468 cells after incubation with carbon dots at excitation wavelengths of (a) at 330-385 nm, (b) 450−480 nm, and (c) 510−550 nm excitation wavelengths, without bright field. The concentration of the CQDs is 55 mg mL-1.

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