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. 2020 Nov 18;10(12):540. doi: 10.1007/s13205-020-02518-5

Synthesis, characterization and biocompatibility studies of carbon quantum dots from Phoenix dactylifera

Kanchanlata Tungare 1,✉, Mustansir Bhori 1, Kavya Sri Racherla 1, Siddhi Sawant 1
PMCID: PMC7674538  PMID: 33240743

Abstract

In the present study, Carbon Quantum Dots (CQDs) were synthesized from Phoenix dactylifera (Date palm fruit) using microwave-assisted pyrolysis and were characterized for its various properties. The synthesized CQD sample exhibited a narrow absorbance peak at 270 nm in UV–Vis spectrum that indicated generation of narrow sized particles. The FTIR analysis of the crude CQDs and dialysed sample revealed the various functional groups involved in the formation of CQDs. TEM data revealed the nature of CQDs to be quasi-spherical and spatially distributed. Biocompatibility of the CQDs was studied using various model systems. CQDs displayed no cytotoxic and anti-clonogenic property when exposed to WRL-68 cell line whereas a slight toxicity was evident in HT1080 post 24 h of incubation suggesting the tremendous potential of the CQDs in the synergistic killing of cancer cells. Phytotoxicity assessment in four different seedlings revealed the non-toxic nature of CQDs. Further these CQDs were found to possess high biocompatibility imposing no inhibition in microbial growth and zilch effect on the development of zebrafish embryos. Thus these CQDs can find immense potential applications in fields of biomedicine as biomolecule detection, drug carriers, fluorescent tracers and in controlling the drug release.

Electronic supplementary material

The online version of this article (10.1007/s13205-020-02518-5) contains supplementary material, which is available to authorized users.

Keywords: Phoenix dactylifera, Carbon quantum dots, Fluorescence, Cytotoxicity, Phytotoxicity, Zebra fish

Introduction

Nanotechnology has evolved as an emerging interdisciplinary field which has found applications in cosmetics, food packaging, healthcare and other industries (Bajpai et al. 2018). The phenomenon of “Nano” is a fascinating concept to study because the properties exhibited by the materials at the nano level are not evident in their bulk forms. One of the most promising fields of research in nanotechnology is nanoparticles. Nanoparticles (1–100 nm size) display unique size-dependent properties such as high surface area to volume ratio, quantum confinement effect, superparamagnetism and surface plasmon resonance (Singh et al. 2011; Khan et al. 2017; Mody et al. 2010).

Recent years have paid much attention to the photoluminescent quantum dots because of their potential use as biological labels, bioimaging agents, drug delivery carriers, biosensors, photocatalysts and optoelectronic devices (Iravani et al. 2020). However, the biological toxicity of these semiconductor Quantum Dots has led to the pursuit of a new environment-friendly alternative that can be used biologically especially in the field of biomedicine (Raj et al. 2012; Chaudhry et al. 2008; Patel and Nanda 2015). Carbon Quantum Dots (CQDs) are nano-sized quasi-spherical carbon particles with a size in nanoscale and have emerged as a fluorescent nanomaterial which can be used to substitute the toxic Quantum dots (Das and Snee 2016). They are superior in terms of their high aqueous solubility, easy availability, chemical inertness, resistance to photobleaching, low toxicity, high biocompatibility, easy functionalization and broad range excitation-dependent fluorescence (Zhang 2014; Wang 2014; Cayuela et al. 2016). Thus, in the past many methods have been devised for efficient synthesis of CQDs like the electrochemical oxidation, ultrasonication, combustion, laser ablation, arc discharge, hydrothermal method and microwave-assisted heating (Tajik et al. 2020).

Of these techniques, microwave-assisted heating induces the interaction of dipole moment of polar molecules in the solvent with alternating electric and magnetic fields leading to molecular-level heating. This technique is advantageous since the high microwave energy generated through the electromagnetic field is directly without any physical contact or medium making the reaction faster with high selectivity yield and energy. Thus the shorter reaction times, environmental friendliness, an energy-saving technique, low impurity, control on size and temperature with improved safety, better reproducibility and great control over experimental parameters results in outstanding preparation of nanoparticles as compared to other conventional heating techniques (Singh et al. 2019).

Albeit several methods report effective synthesis of CQDs, still challenges exist in terms of cost, multi-step procedures as well as formation of toxic by-products (Wang 2014; Cayuela et al. 2016; Dubey et al. 2014). Therefore, it has become exceedingly important to synthesize CQDs from a low cost carbon source which is eco-friendly.

In the current research investigation, we used Date palm fruit as a precursor for CQD synthesis using microwave-assisted pyrolysis method as it is a rich source of carbohydrates having C,H and O elements to support the formation of CQDs by providing an abundance of surface functional groups. The oxygen as a functional group may also additionally provide them with high water solubility and fluorescence properties. Date palm fruit as a natural carbon precursor may exhibit the presence of heteroatoms that can provide good quantum yield and photoluminescence properties. There are plethora of reports on the synthesis of CQDs using natural sources such as banana juice, orange juice, citric acid, lignin, egg-shell membrane, chitosan, gelatin and sugarcane (De and Karak 2013; Sahu et al. 2012; Zhou et al. 2015; Chen et al. 2016; Wang et al. 2012; Yang et al. 2012; Liang et al. 2013; Mehta et al. 2014) but the complexity of natural sources make it difficult to obtain CQDs with satisfactory optical and physiochemical properties. Hence, it is still a challenge and exploratory field to find a good natural precursor with desirable properties. This makes the exploration of more natural carbon precursors and the expansion of their applications highly desirable (Zulfajri et al. 2019). Although Date palm fruit is reported to possess anti-inflammatory, anti-tumor, anti-oxidant, anti-microbial, anti-diabetic and nephroprotective properties (Rahmani et al. 2014; Baliga et al. 2011), no reports on CQD synthesis using this medicinal plant exists to the best of our knowledge. Since the release of nanoparticles into the environment is viewed as a potential threat to the environment and biocompatible nanoparticles pave a path towards exploring the potential of CQDs in numerous applications, we have investigated effects of characterized CQDs for any possible toxicity using different in vitro model systems.

Materials and methods

Chemicals and materials

Seeded Date palm fruits (Falcon, Aqaba Dates) were bought from a local store at Thane, Maharashtra. Fetal Bovine Serum (FBS), Penicillin–streptomycin antibiotic solution, 1 mM sodium pyruvate solution was procured from Hyclone, Thermoscientific, USA. 1X PBS and Dulbecco’s Minimum Essential Medium (DMEM, Catalogue No. CC3009.05L) was obtained from Cell clone, Genetix biotech Asia, India. Gentamicin (50 mg/mL), Trypsin EDTA (0.25% trypsin with 0.2% EDTA in DPBS) and dimethylsulfoxide (DMSO) was obtained from Himedia. All the plasticwares used in animal cell culture were obtained from Nest, India.

Synthesis of CQDs from date palm fruit

CQDs were synthesized using the pyrolysis method in a bottom-up approach. 5 g of whole Date palm fruit were crushed and dissolved in 50 ml of autoclaved distilled water. The solution was filtered using a muslin cloth. Aqueous extract solution was then pyrolysed by heating in a microwave at 100 °C with an output power of 700 Watts for 6 min with 1 ml of 1 N NaOH being added at equal intervals of two minutes. More 50 ml of autoclaved distilled water was added and heating along with NaOH addition was continued for another 6 min. After 12 min of heating, another 50 ml of autoclaved distilled water was added to prepare a brown viscous solution (crude sample). A control sample was prepared by crushing 5 g whole date in 150 ml autoclaved distilled water without any heating.

Purification and separation of CQDs

Purification and separation of CQDs were done by dialysis. Dialysis bags of pore size 2.4 nm (dialysis membrane-150 LA401-1MT) were pre-treated in 50 ml autoclaved distilled water at 40 °C for 1 h. 3 ml of crude solution was added to a dialysis bag and the sample was dialyzed in 100 ml of 1X PBS (pH 7.4). The CQDs were obtained in beaker (dialyzed sample). Further the samples were stored at 4 °C in the refrigerator. All three samples viz. crude, bag and dialyzed samples were subjected to characterization.

Characterization of CQDs

UV–Vis spectroscopy

1 ml of crude sample diluted 1:10 using distilled water was used for UV–Vis spectroscopy (Shimadzu) and the spectrum was recorded in the spectrum mode. The recording range was set from 200 to 1100 nm. 1 ml of 1 X PBS was used as blank and reference. Spectrum was also recorded for 1:10 diluted bag sample and undiluted dialyzed sample.

Fluorescence spectroscopy

For fluorescence studies, fluorescence emission of samples placed in the cuvette was recorded at different excitation wavelengths with an excitation slit width of 5 nm and emission slit width of 10 nm at 400 V PMT voltage. The fluorescence studies were carried out using iControl, Tecan Reader (Germany).

Fourier transform infrared spectroscopy (FTIR)

The FTIR analysis was performed using potassium bromide (KBr) pelleting method. The aqueous CQDs samples were drop-casted onto KBr powder, and ground properly, mixed, and pressed to form a thin pellet. The FTIR recording was performed using 3000 Hyperion Microscope with Vertex 80 FTIR System (Bruker, Germany) between 500 and 4000 cm−1.

Transmission Electron Microscopy (TEM)

The CQDs were further characterized using TEM to gain data regarding structural details like size, shape and spatial arrangement. TEM characterization was carried out at IIT, Bombay. The CQDs samples were diluted in an aqueous solution and drop-casted onto copper grids. The grids were dried for 12 h and imaged under an accelerating voltage of 200 kV.

Lyophilization

To obtain powdered samples, the purified and diluted aqueous CQDs samples were kept in a 50 ml falcon tube with their brim sealed with paraffin (perforated). The frozen samples were then lyophilized for 48 h at − 45 °C. The powdered samples were then stored at ambient temperature for future use.

Biocompatibility and toxicity studies

Cell culture

Authenticated HT1080 and WRL-68 cell lines were procured from National Centre for Cell Science (NCCS), Pune, India. Cells were cultured in DMEM supplemented with 10% heat-inactivated FBS, 1 mM sodium pyruvate and antibiotics (100 U/mL penicillin and 100 µg/mL streptomycin). Cell cultures were maintained at 37 °C in 5% CO2 humidified atmosphere. All experiments were performed on exponentially growing cells with working concentrations 0.1, 1, 10, 100 and 1000 mg/mL.

Phase contrast microscopy

1x104 cells were seeded with 10% DMEM in a 24-well plates. The cells were then treated with CQD solution at various concentrations ranging from 0.1 to 1000 μg/mL for 24 and 48 h. Separate well of control was maintained. Phase-contrast microscopy of cells treated with CQDs and the control was performed using Nikon ECLIPSE TS 100.

Metabolic activity-dependent viability assay

Cell viability was measured using the colorimetric 3-(4,5-dimethylthiazol-2-yl)-2,5- diphenyl tetrazolium bromide (MTT) assay as described by Mosmann (1983). 2500 cells/200 µl were added into each well of a 96 well, flat bottom tissue culture microplate and incubated for 24 h. Cells were then treated with 0.1–1000 μg/mL concentrations of filter-sterilized CQDs for next 24 and 48 h at 37 °C. Control cells with medium only and negative control were also maintained. At the end of the incubation, MTT (20 µl) at the final concentration of 0.5 mg/mL in 1X PBS (pH 7.4) was added to each well and incubated for 4 h at 37 °C in dark. Following incubation, formazan crystals were dissolved by adding 200 µl of DMSO. Absorbance was measured at 570 nm and 655 nm reference wavelength using a microplate reader (Biorad version 680). The cell viability was then calculated and expressed in percentage.

Trypan blue dye exclusion assay for cell viability

Trypan blue viability assay was performed for both cell lines HT1080 and WRL68 as per the protocol described by Strober 1997. 1x104 cells/mL were plated into each well of a six well plate and incubated for 24 h. Cells were then treated with 0.1-1000 μg/mL concentrations of filter-sterilized CQDs for 24 and 48 h with appropriate controls being maintained. Fresh culture medium was exchanged before the addition of CQDs. After incubation, all cells were harvested, mixed with 0.4% trypan blue (9:1) and counted using Neubauer’s improved hemocytometer. Total cell density and percentage viable cells were then calculated.

Clonogenic assay

Clonogenic assay was performed as per the protocol described by Franken et al. 2006. A total of 500 cells were treated with CQDs at 0.1-1000 μg/mL concentrations for 24 h. After the exposure, the media from each well was replaced with fresh medium and cells were maintained under standard culture conditions for the next 7 days. The media was replenished every two days and on the final day media was discarded, cells were washed with 1X PBS followed by fixation with methanol: acetic acid (3:1). Fixed cells were stained using 0.5% crystal violet in methanol for 15 min and survivor cells were photographed using Olympus 1X51 ProgRes Capture Pro 2.7.7 (Jenoptik optical system). Plating efficiency of cells was calculated for all the concentrations.

Antimicrobial activity determination

The antimicrobial studies were performed using agar well-diffusion method. Gram positive (Bacillus subtilis, Cornybacterium dipetheria and Staphylococcus aureus) and gram-negative bacteria (Escherichia coli and Klebsiella pneumoniae) were used as test microorganisms. Culture plates were prepared using “Pour Plate” technique and CQD solution of concentrations ranging from 0.1-1000 µg/mL was added in the wells bored within experimental plates. 0.5 mM Ampicillin was used as positive control and D/W was used as a negative control. The plates were incubated at 37 °C for 24 h and then observed for any possible CQDs mediated antimicrobial effects.

Phytotoxicity analysis

The phytotoxicity study was performed on four plants namely, Mung (Vigna radiata), Pea (Pisum sativum), Wheat (Triticum aestivum) and Corn (Zea mays). The seeds were initially surface sterilized with 1.2% sodium hypochlorite for 1 min and then washed with distilled water for five times. Seeds in experimental plates were soaked with the highest concentration of CQD i.e. 1000 µg/mL and those in the control plate were soaked in distilled water. The germinated seedlings were closely monitored for 7 days and sufficient water was added when required. Root length, shoot length of the germinated seeds was measured till 7th day and percent germination was calculated. The experiment was performed in triplicate for accuracy.

Toxicity studies using zebrafish as a model organism

Zebrafish maintenance and embryo selection

Zebrafish maintenance, culture and embryo selection was done as per Avdesh et al. 2012 protocol. In brief, zebrafish adults with a roughly 2∶1 male/female sex ratios were kept in a 250-L full glass aquarium under the following conditions: 26 ± 1 °C, 14-h/10-h light/dark cycle. Spawning was triggered once the light was turned on in the morning and completed within 30 min. At 4–5 h post-fertilization (hpf), embryos were collected and rinsed several times with the culture medium to remove residues on the egg surface. Healthy embryos at the blastula stage were then selected using a stereo microscope (Labovision 2000) for subsequent experiments.

Fish embryo toxicity test

The fertilized eggs were cultured at a constant temperature at 26 ± 2 °C. The embryos were exposed to CQD at 0.1, 1 and 5 mg/mL concentrations along with the control. The mortality, hatching and heartbeat of the zebrafish larvae was observed at 24, 48, 72 and 96 hpf. Mortality percentages were determined from the total numbers of embryos exposed to CQDs. Mortality, coagulation of embryos, hatching and heartbeat was observed using an inverted microscope (Nikon eclipse, TS100) and stereomicroscope. Each experiment was done in triplicates. The percentage of dead embryos was estimated every 24-h for 4 days. Whereas percent hatching and heartbeat rate were recorded after 48 h. The representative images were captured using a high-definition video camera.

Statistical analysis

Data represents mean ± SD. Statistical analysis was performed using the software “GraphPad Prism”- version 6.01. The significance of difference among the groups was assessed using a one-way analysis of variance (ANOVA) test followed by Tukey’s honest significant difference (HSD) post hoc test of difference between all group means.

Results and discussion

Synthesis

Date palm fruit is rich in carbohydrates (73%), protein (3%), lipid (2.9%), vitamin B complex and mineral elements and is widely consumed all over the world. In the present research investigation, we have synthesized CQDs from Dates palm fruit by microwave-assisted pyrolysis method because it is a rapid, cheap, simple and one-step method for synthesizing CQDs (Rahmani et al. 2014; Baliga et al. 2011). The pyrolysis resulted in the formation of dark brown-colored viscous solution from a yellow-colored solution which gave the first indication towards the formation of CQDs. Purification and separation of CQDs by dialysis yielded light brown solution (Fig. 1a) which displayed green fluorescence when illuminated under UV light.

Fig. 1.

Fig. 1

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Physiochemical properties of CQDs. a Normal daylight and UV illuminated (270 nm) image of as prepared purified CQDs by dialysis along with its absorption spectra, b Fluorescent spectroscopy image of highest fluorescent intensity in the dialyzed sample, c Excitation independent emission of CQDs excited at a different wavelength, d FTIR spectroscopy showing surface functional group rich self-passivized CQDs and (E) TEM analysis displaying the presence of CQDs

Characterization studies of synthesized and purified CQDs

The optical properties of synthesized CQDs were studied using UV–Vis spectrum as illustrated in Fig. 1a. The dialyzed sample showed a narrow absorbance peak at 270 nm. This indicates the presence of narrow sized particles in the dialyzed solution. The peak obtained at 270 nm for dialyzed samples is in accordance with earlier reports by Tyagi et al. 2016 and Linehan et al. 2014. These peaks correspond to n–π* and π–π* transition of the –C=O and C=C bonds of conjugated π-domains. Since the absorption values agree with the peaks of CQDs reported in the literature, the absorbance peaks obtained can be attributed solely to the synthesized CQDs (Pires et al. 2015). An investigation on fluorescence (FL) was carried out using fluorescence spectrophotometer as illustrated in Fig. 1b. When the samples were excited at an excitation wavelength of 250 nm, the highest intensity of fluorescence was recorded at 320 nm for a dialyzed solution and relatively less fluorescence intensity for a crude sample and the contents inside the dialysis bag (bag sample). The dialyzed sample also showed excitation wavelength-independent photoluminescence when excited from 325 nm to 400 nm range with 25 nm increments (Fig. 1c). The photoluminescence peaks did not show any red shifts to longer wavelength when excitation wavelengths were increased. The PL emission ranged from 421 to 460 nm which is violet to blue colored emission, when excitation wavelength was changed from 325 to 400 nm. The intensity of PL emission increased from 325 to 375 nm and decreased for 400 nm. The highest intensity of PL emission was found to be at 445 nm when excited at a wavelength of 375 nm. This may be attributed to their surface state or molecular state of CQDs (Zhai et al. 2012; Yang et al. 2017). Another reason for such excitation-independent fluorescence might be fluorescence resonance energy transfer (FRET). This phenomenon occurs when smaller carbon-dot domains, upon UV excitation emits blue light (high energy) which is in the excitation-range of larger domains, the larger domains will then emit longer wavelength light with lower energy (Fang et al. 2012; Kumawat et al. 2017). This PL emission is different from excitation-dependent PL emission which is mostly observed in CQDs due to presence of surface trap sites, defect states, QCE, surface functional groups and heteroatom doping (Samantara et al. 2016; Zhu et al. 2015).

An IR spectrum gives a molecular fingerprint with each peak corresponding to frequencies of vibrations of each bond present between atoms of the compound. No compound has the same combination of atoms as the other compounds, thus the IR spectra of that compound are its unique characteristic and can be used for qualitative identification of that compound. The width of the peak indicates the number of specific bonds present in that compound thus giving quantitative identification of that compound. FTIR is based on the fact that most molecules absorb light in the infra-red region of the electromagnetic spectrum. This absorption corresponds specifically to the bonds present in the molecule. The frequency ranges are measured as wave numbers typically over the range 4000–1600 cm−1 (Zhang and Cresswell 2016). The FTIR analysis of the crude CQDs and dialysed sample was carried out to determine the functional groups involved in the formation of CQDs (shown in Fig. 1d). FTIR spectra of dialysed samples showed the functional groups which remain in the CQDs after microwave pyrolysis of crude CQD solution. FTIR spectra of crude solution revealed the presence of a broad peak at 3498 cm−1 which corresponds to O–H group with stretching vibrations as well as N–H stretching of secondary amine which falls in 3350–3310 cm−1 range of absorption. Two very sharp peaks were observed at 2922 cm−1 and 2856 cm−1 which corresponds to C-H stretching vibrations of alkanes and O–H stretching vibrations of carboxyl groups. Peaks at 1595 cm−1 represent C=C stretching of the aromatic group, N–H bending vibrations of amide group and 1452 cm−1 represents bending vibrations of C-H of alkanes and C=C stretching of the aromatic group. A very small but broad peak was observed at 1037 cm−1 corresponding to C-O stretching of esters. A small peak is observed at 1626, 1609 and 1231 cm−1 which corresponds to N–H bending and C–N stretching vibrations of the amine group respectively. The functional groups like –OH might have arisen due to moisture content, amino acids like serine and methionine of proteins, sugars like glucose, fructose and sucrose found in dates, vitamin C content, phenolic compounds and flavonoids. The high proportion of C–H groups might have arisen due to the presence of aliphatic chains of carbohydrates, fats and proteins, C=C can be attributed to aromatic structures of vitamin C, phenolic, flavonoid compounds as well as aromatic amino acids such as tyrosine, tryptophan. The –N–H can be attributed to amino acids like asparagine and glutamine while –C–O can be attributed to flavonoid compounds, esters or ethers in dates (Hamad et al. 2015).

Slight differences in peaks were noted in CQDs as compared to the crude because of change in functional groups due to the breaking of old bonds and formation of new bonds during pyrolysis synthesis. The CQDs showed a very sharp peak at 3431 cm−1corresponding to stretching vibrations of the O–H group. Two very small but sharp peaks were obtained at 2925 cm−1 and 2853 cm−1. Both of these peaks are representative of C–H stretching vibrations of alkanes and O–H stretching vibrations of carboxyl groups. A sharp peak was observed at 1626 cm−1 which showed shift to the left when compared to control. This peak corresponds to C=C stretching of the aromatic group, N–H bending vibrations of amide group and a very small, broad peak was observed for 1417 cm−1 corresponding to bending vibrations of C–H of alkanes and C=C stretching of the aromatic group. A very sharp multiple peaks were observed at 1023 cm−1 corresponding to C–O stretching of esters or ethers.

An increase in O–H groups in CQDs (3431.58 cm−1) was prominently seen. This may also be due to addition of 1 N NaOH during synthesis. There was a decrease in the proportion of alkanes and carboxylic groups because of two small peaks obtained (2925 cm−1 and 2853 cm−1). A feeble peak of C=C of the aromatic group (1626 cm−1) was found to be shifted slightly as compared to the corresponding peak of crude (1595 cm−1) indicating the formation of C=C. Increase in proportion of C-O bond of esters was observed in case of CQDs (1023 cm−1) as compared to crude solution. These functional groups might have arisen from the functional groups of the crude sample due to sequential dehydration, polymerization and carbonization of carbohydrates, amino acids of proteins and fatty acids present in Date palm fruit during microwave pyrolysis synthesis (Alam et al. 2015). Functional groups like C–O, O–H and –COOH increase the solubility and hydrophilic properties of CQDs (Pires et al. 2015; Alam et al. 2015). Moreover, they also tune the photoluminescence and promote their applicability in biological systems due to their environment friendliness (Alam et al. 2015).

An investigation of TEM data revealed that the as-prepared CQDs are quasi-spherical in shape which is spatially distributed (Fig. 1e). There is non-uniformity in their size and a narrow-range size distribution ranging from 0.02 to 0.63 µm. The wide variety in size of CQDs is said to be the result of fabrication variability which can also affect the surface functional group and defects (Paulo et al. 2016; Chen et al. 2014).

Effect of CQDs on morphology of cancerous and non-cancerous human cell lines

CQDs find major applications in biomedical fields. Studies have reported that ‘anti- cancer drug loaded CQDs’ not only follow an ideal drug release profile but also show higher killing rate of cancer cells as compared to ‘only drug’ treatment (Mewada et al. 2014). The testing of CQDs against cancerous cell line of HT1080 was carried out with a prospect that availability of anti-cancerous CQDs would be able to further boost the efficacy of these drugs. While CQDs act as efficient target specific drug carriers or fluorescent tracers, their exposure to biological systems makes it mandatory to verify their biocompatibility and hence curtail any of the possible side effects (Lai et al. 2012; Lim et al. 2014). To satisfy this purpose, the CQDs were also tested against non-cancerous mammalian cell line i.e. WRL 68.

Both the cell lines were treated with varying concentration of CQDs ranging from 0.1-1000 µg/mL for 24 and 48 h. Visual observation by phase contrast microscopy revealed no significant changes in morphology at any of the concentrations for any time point (Fig. 2a, b). The cells appeared healthy and no abnormal changes were observed. Control plate showed healthy adherent cells with 70% confluency. No vehicle control was maintained as CQDs stock solution was prepared in complete medium. WRL cell line exhibited normal ****hepatocyte-like epithelial- like morphology even in the highest concentration after 48 h and were unaffected after CQDs exposure. The HT1080 cell line too showed normal epithelial morphology though some amount of floating cells were evident after 48 h. These results exhibit good biocompatibility of CQDs and thus can find application as drug carriers causing no toxicity to the normal cells.

Fig. 2.

Fig. 2

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a Representative phtotomicrograph showing morphology of CQDs treated WRL 68 cells at lowest and highest concentration on different time point (magnification 150X, scale bar=100 µM) a Control (24 h); b 0.1 µg/mL (24 h); c 1000 µg/mL (24 h); d Control (48 h); e 0.1 µg/mL (48 h); f 1000 µg/mL (48 h). b Representative phtotomicrograph showing morphology of CQDs treated HT1080 cells at lowest and highest concentration on different time point (magnification 150X, scale bar=100 µM) a Control (24 h); b 0.1 µg/mL (24 h); c 1000 µg/mL (24 h); d Control (48 h); E: 0.1 µg/mL (48 h); f 1000 µg/mL (48 h)

Studies on CQDs induced cytotoxicity in cell lines

We investigated the repercussion of CQDs on WRL 68 and HT 1080 cell line after two-time point i.e. 24 h and 48 h by MTT and trypan blue dye exclusion assay. The principle of MTT assay relies on the cellular reduction of MTT to a purple formazan product by mitochondrial dehydrogenases of viable cells. The intensity of the purple color formed by this procedure is proportional to cell viability (Mosmann 1983; Strober 1997). Whereas dye exclusion assay is based on the principle that live cells possess intact cell membranes that exclude certain dyes like trypan blue.

By both the viability assay, it was revealed that no significant change was caused in WRL-68 cells when exposed to varying concentrations of CQDs ranging from 0.1 to 1000 µg/mL.

Maximum drop in the viability of cells was observed up to 80% in highest concentration i.e. 1000 µg/mL for both the specified time point (Figs. 3a, b, 4a, b). The difference in the viability shown in highest concentration for both the cell lines was found to be non-significant when compared to control indicating excellent biocompatibility of the CQDs. On the other hand, a significant difference in percentage viability was observed in HT1080 cells when compared to control after 48 h treatment though no significant drop in viability was recorded in these cells for the first 24 h (Fig. 3c, d, 4c, d). Present findings reveal moderate anti-cancerous properties of CQDs when exposed for a longer duration. According to Kollur et al. 2019 the probable mechanism for the anti-cancer properties of CQDs may be attributed to its binding action on the receptors such as nuclear antigen or tumor cell surface antigen that are solely present on the tumor cell membrane to bring about cancer cell disruption (Kollur et al. 2019). CQDs are also previously reported to cause activation of caspase -3 thereby inducing apoptosis in cancer cells and thus are known to be cytotoxic and apoptogenic in nature (Arkan et al. 2018). Thus these CQDs can find tremendous potential when tagged with anti-neoplastic agents to kill cancer cells synergistically and allow simultaneous cell imaging due to its fluorescence. The results obtained are concurrent with the results of previous studies indicating cytotoxicity of CQDs only at very high concentrations (Bhaisare et al. 2015; Ray et al. 2009).

Fig. 3.

Fig. 3

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Effect of CQDs on the viability of cells at different concentrations using dye exclusion method. a WRL-68, 24 h b WRL-68, 48 h, c HT1080, 24 h and d HT1080, 48 h Data represents Mean ± SD values. Symbols in the figure indicates, C Control, NS Non-significant, *p < 0.05; ***p < 0.001

Fig. 4.

Fig. 4

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Effect of CQDs on viability of cells at different concentrations using MTT assay. a WRL-68, 24 h b WRL-68, 48 h c HT1080, 24 h and d HT1080, 48 h. Data represents Mean ± SD values. Symbols in the figure indicates, C: Control; NS Non-significant; *p < 0.05; ***p < 0.001

Effect of CQDs on clonogenic property of WRL-68 cells

Clonogenic assay is a type of survival assays, which tests long-term renewal of cells after treatment of experimental samples. Clonogenic assay was carried out to relatively study reproductive viability of cells, before and after the CQD treatment (Rafehi et al. 2011; Yang 2012). A colony is defined as a cluster of least cells, which can often only be determined microscopically. 500 cells were treated with CQDs at different concentrations for 24 and 48 h. Control cells were also maintained throughout the experiment. After 7 days post plating (dpp), the colony was counted and plating efficiency was calculated. Treatment with CQDs did not alter the plating efficiency of cells significantly when compared to control for all the concentrations at both the time points reflecting CQDs has no cytotoxic and anti-clonogenic property on normal cells which is requisite for theranostic approach (Fig. 5).

Fig. 5.

Fig. 5

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Effect of CQDs on the clonogenic ability of WRL-68 cells after 24 and 48 h. Data represents the value of plating efficiency as Mean ± SD. Symbols in the figure indicate, C Control, NS Non-significant

Antimicrobial activity assessment of CQDs

Microbes are a completely different class of organisms which are expected to have a different result as compared to mammalian cell lines or plants. Studies on microorganisms are needed to check the sensitivity or resistance of microorganisms towards a particular compound since there is continuous rise in the development of resistance for existing antibiotics and hence the need for new antibiotics keeps arising constantly. Also, antimicrobial compounds cannot be released directly into the environment without confirming their safety to microbes which are omnipresent and useful components of nature. Any probable antimicrobial agent with high antimicrobial activity could disturb the balance of the ecosystem. Thus, in the present study we evaluated the consequences of varying concentration of CQDs on the growth of different microorganisms. The control plates display zone of inhibition for positive control and lacks the same for negative control (distilled water). No zone of inhibition or susceptibility was recorded for synthesized CQDs at 0.1, 1, 10, 100, 1000 µg/mL concentration and crude extract for any microorganism, which indicates good biocompatibility of CQDs to both gram-positive as well as gram-negative bacteria (Table 1 and Supplementary Fig. 1). The result of present study is in concurrence with the previously established research data (Mehta et al. 2014).

Table 1.

Anti-microbial activity assessment of CQDs on different microorganisms by agar diffusion method

Concentrations
of CQDs		 Bacillus Subtilis (Gram +ve) Escherichia coli (Gram −ve) Cornybacterium diptheria (Gram +ve) Klebsiella pneumoniae (Gram −ve) Staphylococcus aureus (Gram +ve)

Positive control

(0.5mM Amp)

 + + + + +
0.1 µg/ml − − − − −
1 µg/ml − − − − −
10 µg/ml − − − − −
100 µg/ml − − − − −
1000 µg/ml − − − − −
Negative Control − − − − −
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“−” represents: Absence of susceptibility; “+” represents: Susceptibility ≥ 7mm

Phytotoxicity assessment of CQDs

Seed germination and plant growth bioassays are the most common techniques used to evaluate phytotoxicity of a particular compound (Kapanen and Itavaara 2001). The toxic compounds can affect plant growth causing reduced growth rate, stagnancy in growth and germination or plant death at higher concentration. Few highly toxic compounds have the aptitude of destroying whole farms of plants, thereby investigating the phytotoxicity of the compound before releasing into the environment becomes critical.

Phytotoxicity studies reported no significant changes in either shoot or root length in any of the plant seeds demonstrating excellent phytocompatibility of the CQDs (Fig. 6, Supplementary Fig. 2 and 3). The changes in percentage seed germination of the experimental group were statistically indistinguishable from control. Similar results were reported by Li et al. after exposing Mung seeds with CQDs (Li et al. 2016).

Fig. 6.

Fig. 6

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Bar graphs showing phytotoxicity analysis of CQDs at 1000 µg/mL concentration. a Percent germination of seeds after CQDs treatment in Mung, Pea, Wheat and Corn. Root-shoot length analysis upon exposure of CQDs in b Mung, c Pea, d Wheat and e Corn. The data represents Mean ± SD and NS indicates non-significant results when compared to control

Zebrafish study

The zebrafish embryo is an established in vitro model and is popularly used to estimate the toxicity of various chemicals that may prove harmful to the environment. As per the regulations by the German Institute for Standardization (2001) and International Organization for Standardization (2007), FET with zebrafish (Danio rerio) embryos has become a mandatory component in routine whole chemical testing and was standardized at an international level in 2007. A modified version has been submitted by the German Federal Environment Agency as a draft guideline for an alternative to chemical testing with intact fish (Braunbeck and Lammer 2006). Post fertilization healthy embryos were collected and were exposed to different concentrations of CQDs. Untreated embryo which serves as control were also maintained throughout the experiment. Kaplan- Meier survival curve and morphological analysis showed no significant change in the treated groups when compared to control embryos (Fig. 7a). Percent hatching in developing embryo and heartbeats per minute in larvae were recorded after 48 and 72 h of treatment which was also revealed to be non-significantly different when compared with control (Fig. 7b, c). In the studies conducted by Cheng et al. 2007 no mortality was observed following the exposure to 120, 240, 360 mg/L of single-walled carbon nano-tubes (SWCNTs). There was no significant morphological difference in the developing embryos and hatched larvae after treatment when compared to control (Fig. 8).

Fig. 7.

Fig. 7

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a Kaplan–Meier survival curve in zebrafish embryo after CQD exposure at different concentrations and time points, b Effect of CQDs on percent hatching of embryos after 48 h and c Effect of CQDs on heartbeat per minute of developed larvae after 72 h

Fig. 8.

Fig. 8

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Photomicrograph of early embryonic development of Zebrafish embryo exposed to 5 mg/mL CQDs at 150X magnification. a 6 h of an untreated fertilized egg, b 6 h of a treated fertilized egg, c 24 h of normal development, d 24 h of treated embryos, e 48 h of untreated larvae, and f 48 h of CQDs treated larvae

Conclusion

We have demonstrated a facile and green approach for CQD synthesis from the Date palm fruit and successfully determined the physical characteristics other physical attributes exhibited by the CQDs including size distribution range, particle shape, particle nature and functional groups. All the outcomes of toxicity studies acquired in the present research have uncovered the high level of biocompatibility property possessed by CQDs and has laid a solid base for additional experimentation which could further exploit the imaging property of these CQDs so as to create a huge scope of their applications in bioimaging, drug delivery, biosensor and optronics.

Electronic supplementary material

Below is the link to the electronic supplementary material.

Supplementary material 1 (DOCX 5126 kb) (5MB, docx)

Acknowledgements

Authors gratefully acknowledge Centre for Interdisciplinary Research, D. Y. Patil Deemed to be University, Navi Mumbai for providing seeding funds to the present research work (Project No- CIDR/DYPU/Biotech/013). We would also like to thank Mr. Mukeshchand Thakur and Mr. Mukesh Kumawat, IIT- Bombay, for their help and support through characterization studies.

Author contributions

KT: conceptualization, methodology, software, writing- original draft preparation, reviewing and editing. MB: conceptualization, methodology. KSR: investigation, writing- original draft preparation. SS: investigation, writing- original draft preparation.

Funding

Seeding funds for the project received from Centre for Interdisciplinary Research, D. Y. Patil Deemed to be University, Navi Mumbai (Project No- CIDR/DYPU/Biotech/013).

Availability of data and material

Yes.

Compliance with ethical standards

Conflict of interest

Authors declare no conflict of interest.

Consent for publication

All authors unanimously agree for submission of the manuscript to this journal.

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Associated Data

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Supplementary Materials

Supplementary material 1 (DOCX 5126 kb) (5MB, docx)

Data Availability Statement

Yes.

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