1 Introduction

Nanostructures have attracted attention of scientific and public communities worldwide, and carbon quantum dots (CQDs) have gained remarkable importance owing to their distinct optical, electrical, catalytic, and biological behavior as well as favorable properties such as photo-bleaching resistance, low cost, solubility, extraordinary fluorescence, cell permeability, and good cytocompatibility [1,2,3]. As a result, CQDs have been widely used as substitutes to hazardous metal-based quantum dots and organic dyes in various applications, including cell imaging, sensing, gene transmission, catalysis, drug delivery, and optoelectronics [4]. Xu et al. first discovered CQDs during the purification of single-walled carbon nanotubes in 2004 [5]. Since then, several studies have been conducted to fabricate CQDs by approaches such as thermal decomposition, laser ablation, ultrasonication, plasma treatment, microwave, combustion, arc discharge, electrochemical exfoliation, and hydrothermal oxidation [1, 6,7,8,9,10,11,12]. Studies for the synthesis of CQDs have been carried out using different types of precursor materials such as lampblack, graphite, carbon soot, glycerol, resoles, ethylenediaminetetraacetic acid (EDTA), sucrose, citric acid, l-cysteine, boric acid, glycine, ethylene glycol, glucose, starch, polyethylenimine, benzene, histidine, saccharide, and polyethylene glycol [1, 8, 9, 13,14,15,16,17,18]. However, most CQD precursors are chemicals and expensive. In recent years, environmental benign, cheap, and facile methods have gained popularity for the exploitation of naturally occurring materials as sustainable precursors for CQD synthesis. A few studies have attempted CQD synthesis using renewable and sustainable materials as precursor substances, including edible chicken eggs, milk, ground coffee, water chestnut, apple juice, orange juice, sweet potatoes, garlic, shrimps, grass, orange peel, pineapple peel, oriental plane leaves, lotus leaves, pine needles, leeks, ginkgo leaves, osmanthus leaves, palm leaves, bamboo leaves, Azadirachta indica leaves, Tridax procumbens leaves, Ocimum tenuiflorum leaves, camphor and maple leaf, cotton, oats, lychee seeds, shrimp eggs, peanut skin, coconut milk, date palm fruit, and aloe vera [19,20,21,22,23,24,25,26]. Our previous studies demonstrated that CQDs were derived from sustainable, renewable, and eco-friendly precursors including palmyra palm leaves, fish scale, and dates tree stem [1,2,3]. These studies have encouraged us to fabricate CQDs using naturally available cheap substances as precursors via the green chemistry approach.

Earlier studies have demonstrated the applications of CQDs as fluorescent probes for metal ion detection and cell imaging, owing to their excellent luminescence property. For instance, Li et al. reported the use of citric acid-derived CQDs for imaging of HeLa cells in blue, green, and red fluorescence emission [27]. Huang et al. employed the CQDs derived from sugarcane molasses for the imaging of breast cancer cells and to detect Fe3+ ions [28]. Shrimp egg-based CQDs were shown to easily enter the cytoplasm through the membrane of SK-Hep-1 cells and used for multicolor imaging [19]. Bombyx mori silk-derived CQDs were also found to exhibit good biocompatibility and penetrated the nucleus of A549 cells [10]. According to Zhu et al., the biocompatible and strong green fluorescence of the CQDs derived from graphene oxides may be useful in their application for bio-labeling [29]. Mehta et al. suggested the use of apple juice-based CQDs as molecular probes for microbial cell imaging [30]. Wang et al. also synthesized CQDs from papaya leaf and employed them for Fe3+ ion sensing and Escherichia coli O157 and HeLa cell imaging [31]. Interestingly, Athinarayanan et al. reported that the palmyra palm leaf-derived CQDs employed as fluorescent probes for bio-imaging of human mesenchymal stem cells [2].

Phoenix dactylifera, universally well-known as date palm or date, is an ancient tree that belongs to the Arecaceae family. Mainly, the tree is commonly distributed across the Middle East, Northern Africa, and South Asia regions. In the Kingdom of Saudi Arabia, date palm tree is an important plant, and more than 25 million and 400 varieties of date palm trees are cultivated to obtain approximately 40 kg/tree of agricultural residues [32]. These agricultural residues are improperly utilized. The wastes are dumped in open sites and burnt in open places that may generate greenhouse gases, which may pollute the environment and pose health risks [33]. Hence, it is important to utilize date palm tree residues in a proper way. Although incredible progress has been made in the synthesis of CQDs, there still remains a knowledge gap in this budding field. To the best of our knowledge, no study has explored the use of date palm tree leaves for CQD and hydrochar preparation using integrated process. Herein, we attempt to fabricate CQDs and hydrochar from date palm tree leaves as renewable, sustainable, and environmentally benign precursors using integrated process. Also, we assess the CQDs cytotoxic behavior and use these CQDs as a fluorescent probe for cell imaging applications.

2 Materials and methods

2.1 Materials

The date palm tree leaves were collected from King Saud University campus, Riyadh, Kingdom of Saudi Arabia. Dulbecco’s Modified Eagle’s medium (DMEM), Acridine orange, ethidium bromide, trypsin–EDTA, 3-(4, 5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT), and dimethyl sulfoxide (DMSO) were obtained from Sigma-Aldrich (MO, USA). Fetal bovine serum (FBS) and penicillin–streptomycin were procured from Invitrogen (Carlsbad, California. USA). The cell culture-based experiments were performed using cell culture grade chemicals.

2.2 Fabrication of CQDs

The collected date palm leaves were dried at room temperature and pulverized. The obtained powder was used as a precursor for CQD synthesis. Around 2 g of date palm leaf powder was added to 100 mL of double distilled water. The mixture was transferred to Teflon-lined hydrothermal synthesis autoclave directly kept in an oven at 180 °C for 3 h. Then, the obtained product containing solid and liquid portions was separated by centrifugation. The product was ultracentrifuged for 15 min at 20,000 rpm at 30 °C. The supernatant was carefully separated and filtered by 0.22 µm sterile filter. The liquid portion containing CQDs were dialyzed using 3.5 KD dialysis membrane (Spectrum Laboratories, USA). Then, the CQDs was freeze-dried for further analysis. The obtained solid portion was mixed with 100 mL of double distilled water and transferred to Teflon-lined hydrothermal synthesis autoclave directly kept in an oven at 200 °C for 24 h. Subsequently, the obtained brown color hydrochar was dried for further analysis.

2.3 Analysis of CQDs

The prepared CQD solution was subjected to UV–Vis spectrum analysis between 200 and 700 nm wavelength using Cary 5000 UV–Vis–NIR spectroscopy (Agilent Technologies, USA). The fluorescent behavior of the synthesized CQDs was analyzed with Jasco fluorescence spectroscopy (FP-750). Transmission electron microscopy (TEM) was used to examine the morphological features of CQDs (JEM-1011, JEOL, USA), while phase identification was carried out using an X-ray diffractometer (Rigaku MiniFlex). The surface functional groups of CQDs were analyzed with Fourier transform infrared spectroscopy (FTIR), while their thermal behavior was evaluated with a thermogravimetric analyzer (TGA Q500, TA instruments) following a rise in temperature at a rate of 10 °C/min up to 800 °C. The synthesized CQDs were characterized using atomic force microscopy (Digital Instruments Metrology Group, model MMAFMLN) and the obtained images were analyzed with the WsXm 4.0 software (Nanotec Electronica S. L.) and Gwyddion 2.39. Scanning electron microscopy (SEM) was used to examine the morphological properties of hydrochar (JSM‐7600F, JEOL, USA).

2.4 Cell culture

The hMSCs were obtained from ATCC, USA. The hMSCs were cultivated in Dulbecco’s modified Eagle’s medium (DMEM) supplemented with 1% antibiotics (penicillin/streptomycin) and 10% fetal bovine serum. The cell culture flask was kept in a 5% CO2 incubator at 37 °C.

2.5 Cytotoxicity assessment

The cytotoxicity of date palm leaf-derived CQDs was evaluated using the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay. The influence of CQDs on cell viability was studied using hMSCs as an in vitro model. hMSCs were seeded in a 96-well plate at a density of 1 × 104 cells/well and incubated overnight. The hMSCs were treated with 12.5, 25, 50, 100, and 200 µg/mL of the synthesized CQDs for 24 and 48 h, followed by the addition of 20 μL of MTT dye (5 mg/mL) per well. The plate was covered with an aluminum foil and kept in CO2 incubator for 6 h. The media were carefully removed and the formazan products were dissolved in 200 µL of dimethyl sulfoxide (99.7%). The intensity of the colored product was monitored using a microplate reader at 570 nm (measurement) and 630 nm (reference). The received data were used to calculate percentage cell viability.

$$\mathrm{Cell}\; \mathrm{viability}\left(\%\right)=\frac{\mathrm{Mean\; OD\; of\; treated\; cells}}{\mathrm{Mean}\; \mathrm{OD}\; \mathrm{of}\; \mathrm{untreated\; cells} \left(\mathrm{control}\right)}\times 100$$

2.6 Influence of CQDs on cellular and nuclear morphology

About 30,000 cells/well plated in 24-well plates were incubated overnight. The cells were treated with 25, and 100 µg/mL of CQDs for 24 and 48 h. Following incubation, the media were carefully discarded and the cells were washed using phosphate-buffered saline (PBS). Subsequently, the cells were examined under a bright-field microscope.

For nuclear morphology analysis, the treated cells were stained with acridine orange (AO)/ethidium bromide (EB) dual stain (100 µg/mL of AO and EB, 1:1 ratio). The stained cells were examined under a fluorescence microscope (Carl Zeiss, Jena, Germany).

2.7 Imaging of live cells using CQDs

The cells were cultured on glass coverslips in 6-well plates. After reaching 80% confluency, the cells were treated with 25 µg/mL of CQDs for 10 h. The cells were washed twice with PBS and examined under spinning-disk confocal laser scanning microscope system (Carl Zeiss AG, Germany) at different excitation wavelengths (405, 480, and 568 nm).

3 Results and discussion

3.1 Synthesis of CQDs

Date palm tree is a vital plant in Saudi Arabia that generates a large quantity of biomass every year. This plant biomass mostly contains organic substances, including lignin, cellulose, and hemicellulose. Date palm leaves contain 47% cellulose, 16% hemicellulose, and 36% lignin [34], which attracted our attention. The leaves may serve as precursors for carbon nanostructures. Thus, in the present study, we exploited date palm leaves for CQD synthesis via hydrothermal method. During the hydrothermal treatment process, the date palm leaves were carbonized to form CQDs. Figure 1 describes the mechanism and process of CQD and hydrochar fabrication. The date palm leaves contain proteins and carbohydrates were transformed into CQDs under the pressurized condition at 180 °C.

Fig. 1
figure 1

Schematic presentation of CQD and hydrochar fabrication using date palm tree leaf as a sustainable carbon source for bio-imaging

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Initially, the carbohydrates and proteins were released from date palm leaves biomass. After that, these substances were carbonized to CQDs. To purification of CQDs, the obtained liquid portion from the hydrothermal reaction was centrifuged and dialyzed. Similarly, earlier study demonstrated that palmyra palm biomass was employed as a renewable and eco-friendly precursor for preparing of CQDs [2]. The remaining solid residues were transformed into hydrochar using hydrothermal treatment at 200 °C for 24 h.

The date palm leaf-derived CQDs UV spectra are shown in Fig. 2. CQD spectra exhibited a band at 279 nm owing to π → π* transition of electrons assigned to carbon nanostructures [35, 36]. Figure 2a inset shows the digital photograph of CQDs. After exposure to day light, CQDs turned brown in color but displayed a blue color under UV light (365 nm). Our study results strongly matched with previous studies where various renewable and eco-friendly precursors were employed for CQDs fabrication [1,2,3]. Generally, CQDs are synthesized using different methods, including ultrasonication, electrochemical, and microwave. These CQDs were exhibited a UV–Vis absorption band between 260 and 320 nm, where absorption bands at longer wavelengths are generally because of surface passivation [1, 2].

Fig. 2
figure 2

UV–Vis absorption spectra of date palm tree leaf-derived CQDs (inset figure: CQDs under day light (a) and UV light (b))

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The fluorescence spectra of CQDs are shown in Fig. 3. After increasing the excitation wavelength from 280 to 550 nm, the emission peak slightly shifted toward higher wavelengths and was associated with a decline in fluorescence intensity. Previous studies have reported the bathochromic shift of emission for CQDs [36, 37]. The fluorescence generation mechanism of CQDs is still unknown; however, the strong fluorescence emitted by CQDs is thought to be associated with surface passivation and quantum confinement effects [36]. Our study results revealed that date palm leaves obtained CQDs showed the excitation wavelength-based emission due to the presence of surface emissive trap states and a different size of nanoparticles [38].

Fig. 3
figure 3

Analysis of fluorescence behavior of date palm tree leaf-derived CQDs at various excitation wavelengths

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Figure 4a–b shows the TEM images of date palm leaf-derived CQDs. TEM images clearly revealed that CQDs were well dispersed and spherical in shape, and had a dimeter of 5–15 nm. Atomic force microscopic images revealed the size of CQDs to be 5–15 nm (Fig. 5a–b), consistent with the results of TEM.

Fig. 4
figure 4

a–b Transmission electron microscopy images of date palm tree leaf-derived CQDs at different magnifications

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Fig. 5
figure 5

Date palm leaf-derived CQDs. a Atomic force microscopy image and b height profile

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The morphological features of date palm leaf-derived hydrochar were analyzed using a scanning electron microscope. Figure 6a–b shows the SEM images of hydrochar. SEM images confirmed that hydrochar has a spherical shape with 0.5–2 µm diameter.

Fig. 6
figure 6

a–b Scanning electron microscopy images of date palm leaf-derived hydrochar at different magnifications

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Figure 7a displays the FTIR spectrum of the synthesized CQDs, showing a broad absorption band between 3,500 and 3,400 cm−1, which is related to the O–H group stretching vibration. We also observed bands around 2,950 and 1,410 cm−1 corresponding to C–H and C–N stretching vibrations, respectively. The band at 1,650 and 1,050 cm−1 was assigned to C-O stretching vibrations, indicative of the partial oxidation of CQD surface [2, 39, 40].

Fig. 7
figure 7

a FTIR spectra and b XRD pattern of CQDs

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The X-ray diffraction (XRD) image of date palm tree leaf-derived CQDs is shown in Fig. 7b. The XRD spectrum revealed a single broad peak around 21.2°, owing to the interlayer lattice spacing of ∼3.68 Å. The d value of CQDs was higher than that of bulk graphite (3.34 Å). Accordingly, the synthesized CQDs possessed a turbostratic carbon structure with several functional groups [41]. Earlier studies have reported similar d values for chitosan- and ethanolamine-derived CQDs [42, 43].

Thermogravimetric analysis (TGA and DTG) is broadly used to assess nanostructures thermal stability and surface functionalization of depending on weight loss or gained. Generally, a TGA curve is divided into different stages of decomposition: (i) the initial stage is associated with water or moisture release and (ii) the consequent stages are related to the disintegration of the structure and finally, incineration of the residual material. The thermogravimetric curve of date palm leaf-derived CQDs is shown in Fig. 8a. We observed around 9% weight loss at temperatures up to 200 °C, owing to the removal of moisture. At 300 °C, we found 30% of weight loss due to initiation of surface functional groups denaturation. Approximately 50% weight loss was noted at 500 °C because of the degradation of the organic functional groups on CQDs. We also noticed an around 30% weight loss at 500–600 °C, possibly owing to the release of pyrogas. However, no significant weight loss was observed between 600 and 800 °C. The TGA curve-associated DTG curve (Fig. 8b) displays the peak around 200 °C ascribed to the elimination of moisture, whereas the peaks at 300 °C and 550 °C were corresponding to the thermal decomposition of surface functional groups.

Fig. 8
figure 8

a Thermogravimetric curve and b DTG curves of date palm leaf-derived CQDs

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3.2 Cytotoxic properties of CQDs

The excitation-dependent fluorescence of the date palm-derived CQDs could be used for cell imaging applications. Previously, few studies suggested that CQDs are non-toxic and employed as different biological applications [1,2,3]. Cytocompatibility is an important and essential property that determines the biomedical applications of materials [44,45,46,47,48,49,50,51,52]. We evaluated the cytocompatibility of date palm tree leaf-derived CQDs using hMSCs as a model. The cell viability assay results are shown in Fig. 9. These results suggest that CQDs failed to remarkably alter the viability of hMSCs. Even at 200 µg/mL concentration, the viability decreased only by 1–8%, which clearly demonstrates the biocompatibility of CQDs.

Fig. 9
figure 9

Influence of CQDs on the viability of human mesenchymal stem cells after 24 and 48 h. The data represent mean ± SD of triplicates

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Furthermore, we examined the influence of CQDs on the nuclear morphology of hMSCs using AO/EB staining (Fig. 10). We observed that the cells exposed to CQDs showed green healthy and intact nuclei. The dual staining assay results strongly matched with the cell viability result. Earlier studies reported that non-toxic and cytocompatible nanostructured materials do not change the morphology of nucleus [1,2,3, 44,45,46,47,48,49,50,51,52]. Overall, our study results suggest that CQDs are non-toxic, biocompatible, fluorescent, and benign materials and may exhibit excellent functions in the biomedicine sector.

Fig. 10
figure 10

Effect of CQDs on the nuclear morphology of human mesenchymal stem cells using AO/EB dual staining after 24 and 48 h

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3.3 CQDs as fluorescent probes

We studied the labeling potential of CQDs in hMSCs with confocal laser scanning microscopy. Figure 11 shows that CQDs penetrated into the cytoplasm through the membrane of hMSCs and emitted red, green, and blue fluorescence upon excitation at 568, 480, and 405 nm. As similar, CQDs derived from different precursors were employed as a fluorescent probe [1,2,3]. Overall, we confirmed that CQDs exhibit good fluorescence and are non-toxic materials for biomedical applications.

Fig. 11
figure 11

Imaging of human mesenchymal stem cells using CQDs as fluorescent probes. The cells were examined under a confocal laser fluorescence microscope at different filters. a 405 nm, b 480 nm, c 568 nm, and d overlay of 405, 480, and 568 nm images

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4 Conclusion

The widely available, eco-friendly, and cost-effective date palm tree leaves have been used as sustainable bio-precursors for CQDs and hydrochar synthesis. The date palm leaf-derived CQDs have 5–15 nm in diameter. The hydrochar has spherical shape with 0.5–2 µm in size. The fabricated CQDs showed excellent fluorescent behavior. Also, the CQDs exhibited good biocompatibility even at 200 μg/mL. The cell imaging results revealed that CQDs outstanding performance in the label of cells. Overall, our results conclude that date palm leaf-derived CQDs can be employed for biological applications.