Hydrothermal synthesis of carbon nanodots from wine cork and biocompatible fluorescent probe

We presented a low-cost and simple method to synthesize carbon nanodots (CDs) from waste wine cork using hydrothermal synthesis. The structural and optical properties of the CDs are characterized by TEM, FTIR, Raman, UV-Vis absorption, and photoluminescence (PL) spectra. The analysis results indicated the average diameter of CDs ⁓ 6.2 ± 2.7 nm. Optical measurements showed the phenomenon of excitation-dependent PL and the formation of functional groups on the surface of the


Introduction
Recent nanostructure fabrication methods have allowed researchers to synthesize materials with revolutionary potential applications [1][2][3][4]. There are multiple ways to prepare substances at the nanoscale based on the proposal whereby it can be divided into two routes, bottom-up and top-down [5][6][7][8][9][10][11][12]. The development of fabrication techniques offers many advantages. At the same time, it also prompts scientists to search for novel nanoscale materials, overcoming the limitations of conventional ones.
In terms of carbon nanomaterials, the discovery of carbon nanodots (CDs) should be a case. Although CDs were accidentally discovered by Xu et al when they purified single-walled carbon nanotubes, CDs constitute an active area of fluorescent materials, driven by fundamental questions related to many intriguing optical features [13]. 3 CDs have been defined as carbon nanoparticles with sizes below 10 nm [14,15]. CDs are specifically novel kinds of nanoparticles possessing intrinsically optical properties, which have been associated with high biocompatibility, excellent photostability, as well as the phenomenon of excitation-dependent photoluminescence [16][17][18][19][20][21]. Hence, in recent years, numerous research groups have reported a broad range of applications of CDs including biological labeling, drug delivery, chemical and biosensors, bioimaging, electrocatalysis, etc [22][23][24][25]. For biomedical applications, CDs are considered as a potential solution of future expectation of fluorescent nanomaterials since it takes advantage of traditional semiconductor quantum dots together with their unique characteristic of likely lower cytotoxicity [20,26,27].
For synthesizing CDs, a green and simple preparation is highly desired to overcome the limitation of complex synthesis routes, the involvement of toxic, or expensive reagents. In recent years, the hydrothermal method is considered a cost-effective chemical route for the conversion of carbonization [28][29][30][31][32]. The technique can achieve carbonaceous materials from widely available precursors. Not only the chemical reagents but also the natural sources are favorable to this treatment. Previous works have addressed green biomass precursors could provide an ideal strategy to prepare green fluorescent CDs, especially the waste materials [33][34][35][36][37].
Herein is described the preparation of CDs from waste wine cork (made by cork oak) by hydrothermal treatment. The rationale for the selection of wine cork is considered concerning the polysaccharides content of cork up to 20.9% in the chemical composition [38]. As previously published, many preparations of the polysaccharidesrich material were successfully employed to synthesize CDs [39]. To access the characterization of the obtained CDs, we investigated using analytical techniques, including TEM, FTIR, UV-Vis absorption, and photoluminescence (PL) spectra.
Furthermore, we attempted to probe biocompatibility by incubating mesenchymal stem 4 cells (MSCs) from human umbilical cord (UC) with the obtained CDs. It would provide us information in an adjustment of the CDs synthesized from waste wine cork for bioimaging applications.

Results and Discussion
From the photo presented in Figure 1a, we can see that the aqueous solution containing obtained CDs shows the visible green color under laser irradiation of 405 nm wavelength. In comparison to that under room light, this evidence confirms the formation of fluorescent production to be seen with the naked eyes. As can be seen from Figure 1b, the TEM indeed indicated the obtained particles have a uniform dispersion without obvious aggregation. Specifically, the size histogram of particles presented in Figure 1c indicated the average diameter of CDs ⁓ 6.2 ± 2.7 nm and the value was comparable to the previous report [35,36]. The phase of the obtained CDs was clarified by X-ray diffraction (XRD) pattern and Raman spectra. The XRD shown in Figure 2a illustrated a broad peak at 2θ~22 o and this should be consistent with the (002) lattice spacing, a formation of highly amorphous carbon-based materials [18]. Moreover, the Raman spectra show an intense peak and a shoulder at around 1370 and 1570 cm -1 , which confirmed the existence of both D (sp 3 hybridized carbon atoms) and G (vibrations of sp 2 hybridized carbon atoms) bands [40]. The result obtained in Figure 2b further proves the amorphous nature of the wine cork-derived CDs [40,41].  The optical properties of CDs were investigated by UV-vis absorption and emission spectra. CDs with a quantum yield of 1.54% was calculated using quinine sulfate as 6 reference. The UV-vis absorption spectrum shown in Figure 3a indicates two peaks around 217 and 280 nm. Similar UV-vis spectrum were reported in previous publications, these are attributed to   transition of the CC and n  transition of the CO bonds, respectively [40]. To further clarify optical behavior, we examined fluorescence spectra of the obtained CDs. In early studies, CDs exhibits the dependence of the emission on the excitation wavelength [36,40]. Similarly, in our case, there is an obvious shift as the excitation wavelength varies, as shown in Figure   3b. Particularly, the corresponding contour shown in Figure 3c [42]. The shorter emission band was attributed to the core state emission, whereas the longer one resulted from the surface state emission. More recently, it has been found that fluorescent CDs synthesized from tofu wastewater also presented this effect, in which high and low energy bands have peaks at about 450 and 490 nm, respectively [43]. Up to now, the physical mechanism for photon-induced light emission from CDs is still unclear. The difficult issue is to estimate the role of carbon core and functional groups to the PL spectra and examination of these effects is still a significant challenge. Researchers have proposed that the possible candidate for the physical origin could be attributed to the optical selection of surface state owing to hybridization of the carbon core and the functional groups [20,44,45].  [36,[46][47][48]. In addition, peaks at 1402 and 1060 cm -1 result from the C-O-C asymmetric and symmetric vibrational stretch [49]. According to the above-mentioned assignments, the FTIR spectrum revealed the functional groups on the surface of the carbon core after the thermal reaction. Since these play a very important role in bioconjugation, this promoted us in further probe bioimaging of the obtained CDs.  Meanwhile, the control sample with no treatment of the CDs showed no photoluminescence at the same exposure conditions (data not shown). The results are consistent with the previous publications [19,50,51]. The excitation-dependent PL made it possible for multiple color emission in cells imaged by using CDs only. A possible mechanism of CDs uptake by MSCs is not fully understood and still needs future effort to explain. Up to now, the possible origin could result from the endocytosis mechanism [26,34].

Conclusion
In this study, we successfully synthesized multi colorful fluorescent carbon dots from waste natural precursors using hydrothermal synthesis. The wine cork-derived CDs

Synthesis of CDs
The waste wine cork (5.0 g) was first washed with water to remove surface dirt and dried in an oven before cutting into small pieces. Then, these were mixed with 80 mL of distilled water to transfer into a 100 mL Teflon-lined autoclave. The resulting mixture was then heated at 220 o C for a period of 4 h in an oven. After cooling down to room temperature naturally, the brown-black carbonized solution was roughly purified through a 0.22 μm microporous membrane. Subsequently, the solution was centrifuged at 14000 rpm for 15 min to remove the large particles. Finally, the obtained CDs were stored at 4 o C for further use.

Quantum yield (QY) measurements
CDs with a quantum yield of 1.54% was calculated using quinine sulfate as reference.
The quantum yield of the CDs was determined by a comparative method in which Where m is the slope, n is the refractive index of solvent, and the subscript "r" refers to the referenced sample.

Instrument
We investigated the structural characterization of the obtained CDs by carrying out X-