*Article* **Self-Doped Carbon Dots Decorated TiO2 Nanorods: A Novel Synthesis Route for Enhanced Photoelectrochemical Water Splitting**

**Chau Thi Thanh Thuy 1, Gyuho Shin 1, Lee Jieun 1, Hyung Do Kim 2, Ganesh Koyyada 1,\* and Jae Hong Kim 1,\***


**Abstract:** Herein, we have successfully prepared self-doped carbon dots with nitrogen elements (NCD) in a simple one-pot hydrothermal carbonization method, using L-histidine as a new precursor. The effect of as-prepared carbon dots was studied for photoelectrochemical (PEC) water splitting by decorating NCDs upon TiO2 nanorods systematically by changing the loading time from 2 h to 8 h (TiO2@NCD2h, TiO2@NCD4h, TiO2@NCD6h, and TiO2@NCD8h). The successful decorating of NCDs on TiO2 was confirmed by FE-TEM and Raman spectroscopy. The TiO2@NCD4h has shown a photocurrent density of 2.51 mA.cm−2, 3.4 times higher than the pristine TiO2. Moreover, TiO2@NCD4h exhibited 12% higher applied bias photon-to-current efficiency (ABPE) than the pristine TiO2. The detailed IPCE, Mott–Schottky, and impedance (EIS) analyses have revealed the enhanced light harvesting property, free carrier concentration, charge separation, and transportation upon introduction of the NCDs on TiO2. The obtained results clearly portray the key role of NCDs in improving the PEC performance, providing a new insight into the development of highly competent TiO2 and NCDs based photoanodes for PEC water splitting.

**Keywords:** TiO2 photoanode; L-histidine; nitrogen-doped carbon dots; photoelectrochemical; light harvesting

#### **1. Introduction**

Rapidly spiking global energy demands and pollution caused by the depletion of fossil fuels necessitated the development of natural and renewable sources of energy [1]. Hydrogen is an excellent contender capable of replacing fossil fuels owing to its both eco-friendly and reusable nature. Photoelectrochemical (PEC) water splitting is the most reliable and popular method employed for converting solar light energy into clean and sustainable chemical fuels, such as hydrogen [2,3]. The initial study on photocatalytic water splitting using TiO2 was published way back in 1972 [4]. Since then, different types of semiconductor materials including ZnO, [5] BiVO4, [6] WO3, [7] Fe2O3, [8] SrTiO3, [9] C3N4 [10], and Ta3N [11] were reported as photoelectrodes for PEC. The TiO2 material is considered as the most competent semiconductor for investigating PEC devices due to its characteristics such as advantageous band-edge positions, ease of fabrication, abundance, excellent photocorrosion resistance, eco-friendliness, and cost effective nature [12]. However, application of TiO2 in PEC has been constrained by comparatively greater band gaps for its rutile (3.0 eV) and anatase (3.2 eV) phases [12], severe bulk charge recombination, and slow OER kinetics [13]. As a result, numerous attempts were made to surpass the limitations, such as use of dopants [14], formation of heterojunctions [15], surface modification [16], introduction of defects [17], and quantum dot sensitization [15].

**Citation:** Thanh Thuy, C.T.; Shin, G.; Jieun, L.; Kim, H.D.; Koyyada, G.; Kim, J.H. Self-Doped Carbon Dots Decorated TiO2 Nanorods: A Novel Synthesis Route for Enhanced Photoelectrochemical Water Splitting. *Catalysts* **2022**, *12*, 1281. https:// doi.org/10.3390/catal12101281

Academic Editors: Indra Neel Pulidindi, Archana Deokar and Aharon Gedanken

Received: 26 September 2022 Accepted: 14 October 2022 Published: 20 October 2022

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**Copyright:** © 2022 by the authors. Licensee MDPI, Basel, Switzerland. This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY) license (https:// creativecommons.org/licenses/by/ 4.0/).

Recently, carbon dots (CDs) have been gaining enormous attention by virtue of their fascinating characteristics such as low cost, simple synthesis, functionalization, superior chemical inertness, and photobleaching resistance. Most essentially, CDs are a viable alternative for heavy metal-based QDs and organic dye, owing to its low toxicity with environmental friendliness [18–20]. Since the last decade, astonishing progress has been made in the preparation of CDs either in the top-down or bottom-up route [21,22]. However, new inexpensive, large-scale, and green synthetic approaches of CDs still need to be developed. For instance, a study on the CQDs/BiVO4 and CQDs/NiFe-LDH/BiVO4 demonstrated that after the decoration of CDs on their respective semiconductor, negatively shifted onsite potentials and enhanced charge injection rate were observed in PEC water splitting [22–24]. In addition, CDs, such as CQDs/TiO2 [11] CQDs/ZnO [25], CQDs/WO3 [26], CQDs/BiVO4 [1], and CQDs/bFe2O3 [27], etc., can improve the light harvesting nature of photoanode in ultraviolet region and expand the range of visible region.

The CDs decorated TiO2 films have been reported earlier from different origin materials by different methods and utilized as photoanode for PEC [23]. Zhou et al. utilized glucose as precursor and alkali-assisted ultrasonic chemical method to prepare CDs; and spin-coated TiO2 film with CDs solution [15]. Wang et al. employed a hydrothermal method to synthesize CDs from phloroglucinol [24]. Usually, for photo-driven reactions, N-doped carbon dots exhibit improved activity both theoretically and experimentally than CDs, owing to beneficial quantum confinement and were capable of creating defect-rich heterostructures [25,26]. Based on the N-doping source material, light-harvesting ability and energy levels can be modulated [27], while the functionality of NCDs may interpret the interaction with the semi-conducting material [28]. Han et al. described the process of preparation of N-doped CDs (NCDs) anchored to TiO2 photoanode in electrochemical and hydrothermal methods by using graphite rods and ammonia to obtain a nitrogen-doped CDs (NCDs) solution. This report has demonstrated the enhanced PEC efficiency due to an increased interface charge transfer [12,29]. The report by Wang et al. on NCDs@TiO2 showed an enhanced photocatalytic property owing to its extended light responses with narrowed bandgap upon introduction of NCDs [30]. However, due to the complexity of NCDs with regards to energy states and chemical structure, the mechanism of NCDs in boosting PEC performance remains unknown [25,31]. Moreover, synthesis of CDs and preparation of photoanode was proceeded in multiple steps, which again increases the preparation cost of the electrode [32,33]. Therefore, it is of critical importance to prepare at low cost, as well as understand the nanostructure of NCDs, their interfacial interactions with semiconductor materials and further developments of NCDs.

In the present study, we report the synthesis of new NCDs decorated TiO2 film in a simple one-pot hydrothermal method using L-histidine as source material. The effect of NCDs on TiO2 nanorod film for PEC water splitting has been analyzed systematically by changing the NCDs' loading time from 2 h–8 h. The prepared photoanodes are named as TiO2@NCD2h, TiO2@NCD4h, TiO2@NCD6h, and TiO2@NCD8h. NCDs loaded photoanodes showed higher PEC performance than pristine TiO2, suggesting the contribution of NCDs towards enhancing the performance of PEC. The highest efficiency was found for TiO2@NCD4h (2.51 mA.cm−2), 3.4 times greater than pristine TiO2 (0.73 mA.cm−2). The higher photocurrent for TiO2@NCDs could be ascribed to the improved light harvesting property, decreased rate of recombination, and increased charge carrier density. The detailed characterization of NCDs and NCD loaded TiO2 and PEC water splitting performance analysis were performed and discussed.

#### **2. Results and Discussion**

#### *2.1. Characterization*

FE-SEM and HR-TEM analyses were executed in order to assess the successful loading of NCDs on TiO2 and their morphology and the obtained images are illustrated in Figures 1 and 2. The FE-SEM analysis of pristine TiO2 film (Figure 1a) has shown dense nanorod morphology of TiO2 which have perpendicularly grown on FTO glass showing

an average length of ~2.8 μm and width of ~150 nm. Moreover, no obvious changes in the size and morphology of TiO2 were observed in Figure 1b, even after dipping for 8 h in NCDs solution. Further, HR-TEM (Figure 1c) analysis confirmed the nanorod morphology of TiO2. Moreover, the observed lattice fringes' distance in Figure 1d was 0.35 nm, which corresponds to the d-spacings of the rutile TiO2 (101) planes, which has well-matched with XRD results [12]. Further, HRTEM image of TiO2@NCD4h (Figure 2) showed that NCDs are uniformly loaded on the TiO2 nanorods and appeared in a sphere and ellipsoidal morphology with particle size ranging from 4 to 10 nm. In addition, 0.21 nm lattice spacing was observed for NCDs particle, associated with the (100) facet of NCDs (Figure S3) [15,18,34]. Moreover, to further investigate the distribution of Ti, O, C, and N elements, the elemental mapping analysis was executed, and the respective results are displayed in Figure 2b. The obtained results have shown even distribution of C and N elements on TiO2 nanorods' surface, suggesting the successful decoration of NCDs on the TiO2.

**Figure 1.** (**a**,**b**) Typical SEM of TiO2, TiO2@NCD4h. The corresponding cross-sectional SEM images are shown in the insets. (**c**) FETEM images of TiO2. (**d**) FETEM images of TiO2@NCD4h.

**Figure 2.** (**a1**–**a3**) FETEM of TiO2@NCD4h. (**b**) HAADF-STEM of TiO2@NCD4h and elemental mapping for Ti, O, C, and N.

The crystalline structure of the as-synthesized TiO2 and the effect of NCDs loading time on TiO2 crystallinity (TiO2@NCD2h, TiO2@NCD4h, TiO2@NCD6h, and TiO2@NCD8h) and the orientation growth were examined using XRD analysis. The obtained XRD peaks are displayed in Figure 3. The diffraction peaks of pristine TiO2 films appeared at 36.10◦, 41.27◦, 54.39◦, 62.86◦, and 69.80◦ and correspond to the (101), (111), (211), (002), and (112) crystal planes of tetragonal rutile structure [15,18,35,36]. Moreover, regardless of the loading time of NCDs, the peak positions are the same, but the (101) plane intensity has increased with NCDs loading time. The results suggest that the TiO2 nanorod crystal structure does not get affected by loading NCDs but size of the crystal and preferred orientation directions sparsely get affected. Furthermore, no noticeable diffraction peak of NCDs was observed for TiO2-NCDs, which could be attributed to the modest load of NCDs, lower than the minimum limitation of XRD detection [15,35].

**Figure 3.** XRD patterns of TiO2 and TiO2@NCD2h-8h.

−

As seen in Figure 4, the Raman peaks of TiO2 located at 615.2, 450.5, and 240 cm−<sup>1</sup> correspond to (A1g), (Eg), and multi-photon scattering process, respectively, and represent the TiO2 rutile phase. The peaks which appeared at 1580 and 1333 cm−<sup>1</sup> can be attributed to the D (disordered *sp2*) band and G band of NCDs, respectively [37,38]. Thus, the Raman spectrum of TiO2@NCD4h has shown five peaks, indicating successful fabrication of NCDs in TiO2 nanorods. Furthermore, the enhancement of Raman intensity might be contributed by the increased crystallinity, and it is consistent with the XRD results.

**Figure 4.** Raman spectra of TiO2 and TiO2@NCD4h.

The elemental composition and chemical binding of NCDs decorated on TiO2 catalyst were determined by the XPS analysis (Figure 5). The survey scans illustrated the elements in two structures, e.g., C, N, Ti, and O elements in TiO2@NCDs4h, whereas C, Ti, and O elements were in the pristine TiO2 [12,39] (Figure 5a). An increase in the carbon content and the presence of N1s peak compared with the bare TiO2 evidently confirm that the NCDs were successfully decorated on TiO2. Ti2p spectra (Figure 5b) showed two representative peaks at 464.0 and 458.4 eV (difference: 5.6 eV), which correspond to the spin-orbit coupling for Ti2p1/2and Ti2p3/2, respectively, and was identical to those for TiO2 [15]. Furthermore, Ti2p peaks of the TiO2@NCDs4h structure have considerably shifted (by 0.2 eV) compared to bare TiO2 (Figure 5b). This is due to the electronegativity of C/Ns, which increased the binding ability of extra-nuclear electrons, hence raising the binding energy. The C 1s spectra (Figure 5c) is fitted with three peaks corresponding to C-C, C-O & C-N, and C=O & C=N bonds at 284.8, 286.0, and 288.3 eV, respectively [40]. The N1s spectra (Figure 5d) shows three peaks which appeared at 396.7 eV, 401.4 eV, and 403.7 eV, ascribed to the pyridine-N, pyrrole-N, and Graphitic-N, respectively, indicating the carbon dot doped with the N element [41,42]. In Figure S4, the peak in the O1 s region of TiO2@NCDs@4h was deconvoluted into three peaks at 532.3, 530.1, and 529.6 eV, which are assigned to O-H, C-O, or O-N, and Ti-O bonds in TiO2@NCDs4h, respectively. In agreement with the above microstructure analysis, XPS results suggest that NCDs were successfully deposited on the TiO2 [40,42,43].

**Figure 5.** XPS spectra of pristine TiO2 and TiO2@NCD4h (**a**) survey scan (**b**) Ti 2p (**c**) C 1s (**d**) N 1s.

The absorption properties of the materials are important parameters to estimate the light harvesting nature and energy levels to be used in solar energy conversions. The optical properties of the prepared NCDs in solution and on TiO2 photoanode with changing time were analyzed systematically. The UV-vis spectra of NCDs in solution and TiO2@NCDs thin films are shown in Figure 6a,b and c by changing the loading time. The absorption band of pristine TiO2 ~400 nm represents the rutile TiO2 band edge [44]. After introduction of the NCDs on TiO2, the light harvesting property was enhanced with the increased amount of NCDs loading. The results suggest the successful decoration of NCDs and their contribution in improving the light harvesting property. Moreover, the Tauc plot Equation (S1) was employed to calculate the bandgap (Eg) of pristine TiO2 and TiO2@NCD photoanodes [36,45,46]. The calculated Eg values of TiO2, TiO2@NCD2h, TiO2@NCD4h, TiO2@NCD6h, and TiO2@NCD8h were 3.12, 3.07, 3.03, 3.04, and 3.05 eV, respectively.

**Figure 6.** Absorption spectrum of (**a**) NCDs solutions. (**b**) Pristine TiO2 and TiO2@NCDs thin films (**c**) bandgap energy of Pristine TiO2 and TiO2@NCDs thin films.

#### *2.2. PEC Performance of the Photoanodes*

The effect of newly prepared NCDs on photoelectrochemical water oxidation was studied systematically by changing the loading time of NCDs on TiO2 film and comparing it with the pristine TiO2. Figure 7a shows linear sweep voltammetry performance of TiO2, TiO2@2hNCD, TiO2@4hNCDs, TiO2@6h NCDs, and TiO2@8hNCDs, and their photocurrent data at 1.23 V are illustrated in Table S2. The pristine TiO2 film displayed a 0.73 mA.cm−<sup>2</sup> photocurrent at 1.89 V vs. RHE. After loading NCDs upon the TiO2 film, an enhanced photocurrent was observed compared the pristine TiO2, which instigated to perform optimization studies to improve the NCDs loading and thereby achieve optimum PEC performance using TiO2 with NCDs. In order to optimize the TiO2@NCDs photoanodes, the decorated NCDs on TiO2 was controlled by monitoring the hydrothermal reaction. In Figure 7a, the photocurrents of four NCDs decorated TiO2 (TiO2@NCDs)-based photoanodes showed higher photocurrent than pristine TiO2, indicating the contribution of NCDs in enhancing PEC performance of TiO2. Significantly, the photoanode corresponding to TiO2@NCD2h has displayed an improved photocurrent density of 2.33 mA.cm−<sup>2</sup> at 1.89 V vs. RHE, while pristine TiO2 photoanode has shown 0.73 mA.cm−<sup>2</sup> at 1.89 V vs. RHE. Further, by increasing the loading time from 2 h to 4 h (TiO2@NCD4h), the photocurrent density has also increased to 2.51 mA.cm−<sup>2</sup> at 1.89 V vs. RHE, which was 3.4 times greater than the pristine TiO2. Moreover, TiO2@NCD4h photoanode possesses both enhanced photocurrent density and smaller onset potential than pristine TiO2. The higher photoresponse of NCD decorated TiO2 might be due to the addition of NCD which could effectively promote the separation of photogenerated electron-hole, and promote the capture of water molecules and intermediates in the process of water decomposition

by electrons and holes at the interfaces [15,47,48]. However, further increasing the NCDs loading by increasing loading time to 6 h (TiO2@NCD6h) and 8 h (TiO2@NCD8h) showed declined photocurrent density of 1.99 and 1.85 mA.cm−<sup>2</sup> at 1.89 V vs. RHE, respectively. In addition, onsite potentials also increased compared to the TiO2@NCD4h-based films, possibly due to the variation in conductivity by decorated NCDs. This phenomenon will be further discussed in electrochemical impedance spectroscopy (EIS) section [29].

**Figure 7.** (**a**) Photocurrent density vs. applied potential curves; (**b**) Transient photocurrent density curves at 1.89 V vs. RHE of the as-prepared photoelectrodes; (**c**) Stability test of pristine TiO2 and TiO2@NCD4h at 1.89 V vs. RHE.

Chronoamperometric analysis was performed under chopped illumination for all the prepared electrodes at 1.89 V vs. RHE for 30 s to better understand photo response with time and stability. As depicted in Figure 7b, the photocurrent has rapidly increased immediately after illumination and sharply fell to zero upon stopping the illumination. It confirms that the prepared photoanodes have a well-reproducible photocurrent. Meanwhile, after decorating NCDs on TiO2, the response speed was boosted compared with the pristine TiO2, indicating that the presence of NCDs can significantly reduce the charge recombination in the intersection of electrolyte and photoanode surface. The highest photocurrent density was detected for the TiO2@NCD4h, which is consistent with the observed LSV results. In order to understand the durability of the prepared electrode, 1 h of continuous illuminated chronoamperometric analysis was performed with a high performing photoanode (TiO2@NCD4h) by comparing with the pristine TiO2 based photo anode at same experimental condition. As displayed in Figure 7c, after continuous illumination for 1 h, TiO2@NCD4h has retained 99% of its initial activity, which supports the excellent stability of NCDs decorated TiO2 photo anode. The current densities have matched well with the

LSV data. Both TiO2 and TiO2@NCD4h showed excellent stability after 1 h of continuous irradiation without any photocurrent decay.

The incident photon-to-current conversion efficiency (IPCE) was evaluated to analyze the contribution of various photons in obtaining solar photocurrent. The IPCE has been deduced by using the formula (1):

$$\text{IPCE} \left( \% \right) = \frac{1240 \text{J} (\lambda)}{\lambda P (\lambda)} \times 100 \text{ (\%)} \tag{1}$$

where, *P*(*λ*), *λ*, and *J*(*λ*) are the intensity of a specific wavelength, wavelength of incident light, and photocurrent density at specific wavelength, respectively. Figure 8a shows enhanced IPCE after decorating NCDs on TiO2 with highest IPCE of 29.76% at ~390 nm for TiO2@NCD4h, which was ~3 times greater than the pristine TiO2 (9.76%). The IPCE trend was consistent with the obtained photocurrent density and the enhanced IPCE region is in good agreement with the optical absorption properties. The improved IPCE after the introduction of NCDs to the TiO2 reveals the contribution of NCDs in obtaining enhanced photocurrent density.

**Figure 8.** (**a**). Incident photon-to-current conversion efficiency (IPCE) curves; (**b**) Applied bias ABPE of TiO2, TiO2@NCD2h, TiO2@NCD4h, TiO2@NCD6h, and TiO2@NCD8h measured at 1.23V vs. RHE.

Besides, the applied bias photon-to-current efficiency (ABPE) has been calculated by using the Equation (2):

$$\text{ABPE} \left( \% \right) = \frac{J(1.23 - V)}{P} \times 100 \left( \% \right) \tag{2}$$

where *P*, *J*, and *V* are the power density of incident light (100 mW cm−2), photocurrent density (mA cm−2), and the applied bias (V vs. RHE), respectively. As seen in Figure 8b, the pristine TiO2 reached maximum 0.11% ABPE at 0.88 V vs. RHE, while TiO2@NCD4h reached 1.37% photo conversion efficiency at 0.36 V vs. RHE, 12 times greater than the pristine TiO2 ABPE, suggesting an effective electron-hole pairs separation after the introduction of NCDs [12].

To further comprehend the interfacial charge transfer kinetics at the intersection of photoanode and electrolyte, EIS was employed under illumination and the respective Nyquist plots are displayed in Figure 9a. The decreased radius order of semi-circle was TiO2 > TiO2@NCD8h > TiO2@NCD6h > TiO2@NCD2h > TiO2@NCD4h. The smallest arc of the TiO2@NCD4h compared with its counter parts demonstrates the improved interfacial charge transfer kinetics due to the introduction of NCDs [18]. Furthermore, using Zview software program, the EIS curves have been fitted with an analogous circuit model given in Figure 9a inset, where CPE, Rct, and Rs indicate the constant phase element, charge transfer resistance, and series resistance at the electrolyte/electrode interface, respectively. The observed Rct values of TiO2 and NCDs decorated TiO2 films (2 h–8 h) were 376.0 Ω, 273.6 Ω, 248.3 Ω, 277.2 Ω, and 297.4 Ω, respectively. The lowest Rct value of TiO2@NCD4h further demonstrates the advantage of NCDs decorated TiO2 nanorods in enhancing the charge separation and transfer kinetics.

**Figure 9.** (**a**) EIS spectra; (**b**) 0 Mott–Schottky plots of TiO2, TiO2@NCD2h, TiO2@NCD4h, TiO2@NCD6h, and TiO2@NCD8h. (**c**) Schematic energy levels of TiO2, TiO2@NCD4h.

Mott–Schottky analyses have been executed to estimate the energy band position of pristine TiO2 and TiO2@NCDs, and the corresponding curves are displayed in Figure 9b and the data are depicted in Table S1. The positive slope of both curves indicates n-type semiconductor of TiO2 [18]. The flat band (VFB) potential can be calculated following the Equation (S1). The obtained VFB values of TiO2 and TiO2@NCDs (2 h, 4 h, 6 h, and 8 h) were 0.17, 0.27, 0.34, 0.22, and 0.19 V vs. NHE, respectively, which could be accomplished by the *X*-axis intercept. Moreover, NCDs decorated TiO2 films have shown decreased VFB than pristine TiO2, suggesting an increased band bending of the photoanode, favorable to enhance the charge transfer between the photoanode interfaces and electrolyte. Eventually, enhanced PEC was observed for NCDs decorated TiO2 films. As per the available literature [49,50], the bottom of the conduction band (CB) was −0.1 V lower than the VFB of an n-type semiconductor [50]. Therefore, the CB of TiO2 and TiO2@NCDs were estimated to be positioned at less than 0.1 V of their VFB [33]. Based on the VFB, the CB edge of TiO2 and TiO2@NCDs (2 h, 4 h, 6 h, and 8 h) were determined to be at 0.07, 0.17, 0.24, 0.12, and 0.09 V, respectively. Particularly, doping of NCDs promotes a downward shift in energy levels towards higher potentials and enhances the carrier density in TiO2 [51].

#### **3. Experimental**

#### *3.1. Materials*

All chemicals were used directly without purifying any further. Hydrochloric acid (35%) was obtained from OCI Company Ltd., titanium butoxide (TBOT, 98%) was purchased from Sigma-Aldrich, L-histidine (98%) was procured from Alfa Aesar, and sodium sulfate anhydrous (99%) was obtained from Duksan. For all the experiments, Milli-Q water (MΩ 18) was used.

#### *3.2. Preparation of Rutile TiO2 Film (TiO2):*

The FTO coated glasses (1.5 mm × 2.5 mm, 8 <sup>Ω</sup>/cm2) were cleaned ultrasonically using detergent, milli-Q water, ethanol, and acetone for 1 h, respectively. TiO2 film was synthesized by following a reported hydrothermal method with certain modifications [18]. Under continuous stirring, 0.33 mL titanium (IV) butoxide was added dropwise to 20 mL equal volumes of HCl (35%) and milli-Q water mixed solution until it turned translucent. The solution was then moved to a 50 mL autoclave lined with Teflon, and the FTO glass was placed against the walls of Teflon vessel, conducted side down, for 12 h and heated to 150 ◦C. The TiO2 layer was completely cleaned with milli-Q water and ethanol after cooling to RT, before being sintered in air at 450 ◦C for 1 h.

#### *3.3. Preparation of TiO2@NCDs:*

NCDs have been prepared using a hydrothermal approach (Scheme 1). First, 0.2 g of L-histidine was included in 20 mL mixture of milli-Q water and HCl in the ratio of 19:1. The solution was shifted to 50 mL autoclave, and two TiO2 films on FTO glasses were inserted in the Teflon vessel with the TiO2 side facing down. Then, the hydrothermal reaction was performed at 180 ◦C for 2, 4, 6, 8, and 10 h. The samples were denoted as TiO2@NCD2h, TiO2@NCD4h, TiO2@NCD6h, TiO2@NCD8h, respectively (Figures S1 and S2). The TiO2 @NCD films were extensively washed with milli-Q H2O and ethanol upon cooling to RT. Then, copper wires and as prepared photoanodes were adhered using silver paint. The samples were air dried for 3 h. Finally, the samples were encased by nonconductive epoxy with the illuminated area of 1 cm<sup>2</sup> and left to rest in air for at least 3 h.

**Scheme 1.** Schematic illustration of TiO2@NCDs.

#### **4. Conclusions**

In conclusion, the new NCDs were successfully prepared in a simple one-pot hydrothermal synthesis method using L-histidine as an initial precursor and the as-prepared NCDs were decorated on the TiO2 nanorod-based photoanode. The as-prepared NCDs and NCD decorated TiO2 nanorods were well characterized using FE-SEM, HR-TEM, EDS elemental mapping, which revealed the nanorod morphology of TiO2 and uniform distribution of CDs on TiO2 surface while, XPS and Raman analyses have confirmed the successful self nitrogen element doping, preparation and decorating of NCDs on TiO2 nanorod. The effect of NCDs decorated TiO2 was tested for photoelectrochemical water splitting analysis systematically by changing the loading time of NCDs from 2 h to 8 h. The highest

efficiency was observed for the TiO2@NCD4h-based photoanode (2.51 mA.cm−2), which was a 3.4 times higher photocurrent density than the pristine TiO2-based photoanode. It might be attributed to the increased light harvesting property with charge separation and transportation. The observed IPCE of TiO2@NCD4h has shown 3 times higher quantum yield (29.76%) than pristine TiO2 (9.76). In addition, the calculated ABPE was 12% higher for TiO2@NCD4h than the pristine TiO2, which revealed the enhanced light harvesting property of photoanodes upon loading the NCDs. Moreover, the reduced charge transfer resistance and higher charge carrier density, as observed from EIS and Mott–Schottky analyses, respectively, further support the advantage of newly prepared NCDs in enhancing the PEC performance by promoting effective charge separation and transportation. This study may open up new insights into the rational design and synthesis of highly efficient photoanodes for PEC water splitting.

**Supplementary Materials:** The following supporting information can be downloaded at: https: //www.mdpi.com/article/10.3390/catal12101281/s1; Figure S1: Imagies of NCDs' solution under natural and UV light. Figure S2. NCDs solution based on the reaction time. Figure S3. TEM of NCD. Figure S4. XPS O 1s spectra of TiO2@NCDs4h. Table S1. EIS Data of TiO2 and NCD decorated TiO2 photoanodes Table S2. The photocurrent densities of the TiO2 and TiO2@NCDs photoanodes.

**Author Contributions:** Conceptualization, G.K., C.T.T.T.; Experiments, C.T.T.T., G.S., L.J.; data curation, C.T.T.T., G.K.; formal analysis, H.D.K.; funding acquisition, J.H.K.; supervision, J.H.K. and G.K. writing—review and editing, C.T.T.T., G.K. and J.H.K. All authors have read and agreed to the published version of the manuscript.

**Funding:** This work was supported by the Korea Institute of Energy Technology Evaluation and Planning (KETEP) and the Ministry of Trade, Industry & Energy (MOTIE) of the Republic of Korea (20214000000720). This work was supported by "Human Resources Program in Energy Technology" of the Korea Institute of Energy Technology Evaluation and Planning (KETEP), granted financial resource from the Ministry of Trade, Industry & Energy, Republic of Korea. (No. 20204010600100).

**Data Availability Statement:** The data presented in this study are available upon request from the corresponding author.

**Conflicts of Interest:** The authors declare no conflict of interest.

#### **References**


## *Article* **Eco-Friendly Synthesis of Functionalized Carbon Nanodots from Cashew Nut Skin Waste for Bioimaging**

**Somasundaram Chandra Kishore 1,†, Suguna Perumal 2,†, Raji Atchudan 3,\*,†,**

**Thomas Nesakumar Jebakumar Immanuel Edison 4,†, Ashok K. Sundramoorthy 5,†, Muthulakshmi Alagan 6, Sambasivam Sangaraju <sup>7</sup> and Yong Rok Lee 3,\***


**Abstract:** In this study, *Anacardium occidentale* (*A. occidentale*) nut skin waste (cashew nut skin waste) was used as a raw material to synthesize functionalized carbon nanodots (F-CNDs). *A. occidentale* biomass-derived F-CNDs were synthesized at a low temperature (200 ◦C) using a facile, economical hydrothermal method and subjected to XRD, FESEM, TEM, HRTEM, XPS, Raman Spectroscopy, ATR-FTIR, and Ultraviolet-visible (UV–vis) absorption and fluorescence spectroscopy to determine their structures, chemical compositions, and optical properties. The analysis revealed that dispersed, hydrophilic F-CNDs had a mean diameter of 2.5 nm. XPS and ATR-FTIR showed F-CNDs had a crystalline core and an amorphous surface decorated with –NH2, –COOH, and C=O. In addition, F-CNDs had a quantum yield of 15.5% and exhibited fluorescence with maximum emission at 406 nm when excited at 340 nm. Human colon cancer (HCT-116) cell assays showed that F-CNDs readily penetrated into the cells, had outstanding biocompatibility, high photostability, and minimal toxicity. An MTT assay showed that the viability of HCT-116 cells incubated for 24 h in the presence of F-CNDs (200 μg mL–1) exceeded 95%. Furthermore, when stimulated by filters of three different wavelengths (405, 488, and 555 nm) under a laser scanning confocal microscope, HCT-116 cells containing F-CNDs emitted blue, red, and green, respectively, which suggests F-CNDs might be useful in the biomedical field. Thus, we describe the production of a fluorescent nanoprobe from cashew nut waste potentially suitable for bioimaging applications.

**Keywords:** cashew nut skin; carbon nanodot; human colon cancer cell; cell viability; bioimaging

#### **1. Introduction**

Carbon nanodots (CNDs) [1], carbonized polymer dots [2], carbon quantum dots [3], graphene quantum dots [4], and other nanoscale carbon particles with dimensions of ~≤10 nm are all regarded as carbon dots (CDs) and are considered a new class of fluorescent carbon-based nanomaterials. Xu et al. accidentally discovered CDs in 2004 while purifying carbon nanotubes [5]. Ever since, a wide range of CDs with various chemical and optical properties have been produced using a number of different techniques. The characteristic features of CDs, which include tunable fluorescence emission [6], aqueous dispersibility [7],

**Citation:** Kishore, S.C.; Perumal, S.; Atchudan, R.; Edison, T.N.J.I.; Sundramoorthy, A.K.; Alagan, M.; Sangaraju, S.; Lee, Y.R. Eco-Friendly Synthesis of Functionalized Carbon Nanodots from Cashew Nut Skin Waste for Bioimaging. *Catalysts* **2023**, *13*, 547. https://doi.org/10.3390/ catal13030547

Academic Editors: Indra Neel Pulidindi, Archana Deokar and Aharon Gedanken

Received: 26 December 2022 Revised: 8 March 2023 Accepted: 8 March 2023 Published: 9 March 2023

**Copyright:** © 2023 by the authors. Licensee MDPI, Basel, Switzerland. This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY) license (https:// creativecommons.org/licenses/by/ 4.0/).

chemical inertness [8], biocompatibility [9], and ease of functionalization [10], make them powerful alternative semiconducting nanomaterials. Various biomedical utilities, such as nanoplatforms for biosensors [11], bioimaging [12], drug delivery vehicles [13], and gene transfer [14], are made possible by the ability of CDs to coexist with biological tissues without causing adverse effects. Because of their high fluorescence quantum yields, CDs are used as fluorescent probes in biological samples [15], and can be easily functionalized with biomolecules such as peptides or antibodies. In addition, they have a low photobleaching characteristic nature [16]. CDs are particularly useful for in vivo bioimaging and can be functionalized for targeted drug delivery. Worldwide, one in every six deaths is caused by cancer, which is the second most frequent cause of death. Uncontrolled cell growth is a main characteristic of cancer. For the effective treatment of cancer, early diagnosis is essential. Early cancer detection can help choose the best course of treatment and increase patient survival. Important information about a disease's course and a patient's response to therapy is provided by a diagnosis, which aids in modifying the patient's treatment plan while they are undergoing it [17]. The intriguing physicochemical and optical characteristics of CDs have great potential in the diagnosis and treatment of cancer. Furthermore, CDs can increase the efficacy and delivery of molecules because they are readily absorbed by cells, but more research is required to determine their safety and efficacy for in vivo applications.

Regardless of the synthetic process used to produce nanomaterials, the production of CDs can be categorized as top-down, bottom-up, or physical or chemical. In general, physical techniques involving arc discharge [18], laser ablation [19], and electrochemical etching [20] are hazardous to the environment and difficult to manage. Hydrothermal (HT) [21], ultrasonic [22], microwave-assisted [23], thermal decomposition [24], and electrochemical [25] processes are examples of chemical methods. The HT approach is usually used to produce CDs because it uses mild chemicals and is inexpensive. This approach has been widely employed to prepare a variety of carbon compounds because HT synthesis has negligible toxicological impact. Furthermore, HT conditions can cause reagent solubility, enhance chemical and physical reactions, and enable carbonaceous structures to develop. Conventionally, developing materials with high carbon contents, such as carbon nanotubes, mesoporous carbon, graphene, and graphitic carbon compounds, requires high temperatures (300–800 ◦C), whereas those produced by dehydration and polymerization are produced at lower temperatures (<300 ◦C), and often possess various surface functional groups after carbonization. In general, CNDs can be functionalized and doped with heteroatoms to enhance their fluorescence characteristics and quantum yields. Particularly, the HT method has become more popular for the synthesis of functionalized CNDs (F-CNDs) because it is a one-step procedure without additional oxidation and passivation, has gentle reaction parameters, and requires inexpensive equipment.

CDs are noted for their photophysical characteristics, particularly their fluorescence properties [26], which, like structure, morphology, and composition, are sensitive to the precursors and preparation techniques used [27]. In general, CDs are composed of crystalline carbon cores and decorated with carboxylic acid, alcohol, and amine functional groups [28]. Several biosources, such as lemon juice [29], leaf extract [30], grape juice [31], honey [32], hair [33], carrot juice [34], garlic [35], egg [36], betel leaf [37], and food waste [38] are used as CD precursors.

It is generally known that the transitions between intrinsic states cannot fully account for CD optical properties. The emission of many CDs, however, appears to be primarily influenced by surface-related extrinsic contributions, such as emissions from surface defects and surface charge traps. A proper passivation procedure is essential to produce highly fluorescent CDs, and solvents and pH significantly impact CD fluorescence. Research goals in the engineering area include tuning the photophysical and electrochemical properties of CDs by altering ground and excited state properties [39] and modifying the form, chirality, composition, size, and surface chemistry of CDs [40]. In the present study, we sought to develop non-toxic, <10 nm sized CDs compatible with aqueous environments using *Anacardium occidentale (A. occidentale)* nut skin waste as a precursor for the synthesis of F-CNDs.

Cashew is the popular name for *A. occidentale* (AO), a member of the *Anacardiaceae* family, and it is commonly grown in tropical areas of India, Brazil, and Africa. An essential by-product generated during the processing of cashews is the testa (skin) of the cashew kernel. The resulting testa is a potential candidate for commercial exploitation given that cashew kernels are consumed worldwide on an annual basis in excess of 1,000,000 tonnes. It is said to be an excellent source of hydrolyzable tannins. The cashew nut is a significant cash crop worldwide. India produces and exports the most cashew kernels worldwide, making up nearly 50% of all exports. A brown skin, known as testa, completely envelops cashew nuts, and this skin is one of the best sources of hydrolyzable tannins such as catechin, epicatechin, and epigallocatechin [41,42]. The seed testa has the greatest proportion of phenolic compounds that serve as a barrier of protection for the cotyledon in seeds. Furthermore, it also contains high levels of three phenolic acids, viz. syringic, gallic, and p-coumaric acids [43], which confer significant antioxidant activity [44]. In order to understand the possible mechanisms behind the formation of F-CNDs, it is presumed that the testa of cashew nuts consists of hydrolyzable tannins, phenolic acids, and various other molecules. These constituents undergo the process of dehydration, polymerization, and carbonization to form F-CNDs. We investigated AO biomass-derived F-CNDs synthesized using the HT approach at a lower temperature of 200 ◦C. The best quality F-CNDs with significant fluorescence properties were subjected to cellular imaging of human colon cancer cells.

#### **2. Results and Discussion**

FESEM images of F-CNDs at different magnifications are provided in Figure 1a–c. F-CNDs formed a thin layer over the surface of the sample holder. EDX revealed the elements present on the surface of F-CNDs (Figure 1d–g). Elements were identified by color, e.g., green, red, and yellow indicated carbon (C), oxygen (O), and nitrogen (N), respectively. O and N were distributed evenly over carbon substrates. EDX peaks shown in Figure 1h confirmed the presence of carbon, nitrogen, oxygen, silicon, and platinum. Silicon and platinum were attributed to sample preparation. For FESEM analysis, F-CNDs were spin-coated on silicon wafers and sputtered with platinum.

HRTEM was used to determine F-CND morphology and sizes. The morphological features of F-CNDs are well demonstrated by the micrographs in Figure 2a–c. F-CNDs were observed as spherical, well-dispersed dark dots with a few aggregations. In the high magnification, it is clear that F-CNDs were composed of graphitic layers with an interlayer spacing of 0.21 nm (inset in Figure 2c). The particle size distribution of F-CNDs is shown as a histogram in Figure 2d, which was derived via Gaussian particle-size-distribution fitting and by measuring the sizes of 100 randomly selected particles in HRTEM images (Figure 2a. F-CND sizes ranged from 1.5 to 4 nm with a mean particle size of ~2.5 nm).

X-ray powder diffraction was used to determine the crystal phases in F-CNDs. Figure 3a shows that the XRD spectrum of F-CNDs contained a broad peak at 2θ = 23◦, corresponding to the (0 0 2) carbon lattice [45]. The shoulder peak at 2θ = 43◦ was ascribed to the (1 0 0) plane, and the corresponding d-spacing value was 0.21 nm, which agreed well with TEM results. The absence of a sharp peak, corresponding to the formation of an amorphous layer on F-CNDs, suggested the presence of surface functional groups. F-CNDs were also subjected to Raman spectroscopy to determine the purity and degree of graphitization of samples. The Raman spectrum of F-CNDs is shown in Figure 3b. Two prominent peaks corresponding to carbon D and G bands were observed at 1360 and 1585 cm–1, respectively [46]. These bands correspond to the disorder (vibration of sp3 carbon atom) and graphitic nature (vibration of sp<sup>2</sup> carbon atom) of carbon materials and had an intensity ratio (ID/IG) of 0.63 [47,48], which confirmed a graphitic nature and a few surface defects [48,49]. The deconvoluted Raman spectrum shown in Figure 3c was used to assess the degree of graphitization in F-CNDs. Areas of the D and G bands (AD and AG, respectively) were used to calculate the areal D to G ratio (AD/AG), which was 0.65. This value indicates the formation of graphitized F-CNDs with minimal surface disorder or few defects. Surface disorder could be due to functional groups or edge effects. An

ATR-FTIR (attenuated total reflectance-Fourier transform infrared) spectrum of F-CNDs provided information about surface functional groups (Figure 3d). The hydrophilic nature of the F-CNDs was confirmed by the presence of N–H and O–H stretching vibrations at 3500–3100 cm–1 [50,51]. Peaks between 2870 and 2962 cm–1 were assigned to the C–H asymmetric and symmetric stretch [52]. The presence of carboxyl/carbonyl groups was confirmed by C=O and C=C stretching vibration peaks at 1670 and 1575 cm–1, respectively [53]. The peaks between 1021 and 1120 cm–1 indicated the presence of the C–O–C group, and peaks at 1445, 1260, and 1397 cm–1 were ascribed to–C–N, C–OH and bending vibrations of N–H and O–H, respectively [54]. Out-of-plane stretching vibrations of C–H were confirmed by an absorption band at 665 cm–1 and were attributed to the carboxylic groups on F-CNDs [55]. These findings show that F-CNDs were composed of C, N, and O and decorated with–COOH, –OH, and –C–N groups.

**Figure 1.** (**a**–**c**) FE-SEM images of functionalized carbon nanodots (F-CNDs; (**d**–**g**) EDX elemental mapping images of F-CNDs (**d**) carbon, (**e**) oxygen, (**f**) nitrogen, and (**g**) overlapping image showing all elements; (**h**) EDX spectrum of F-CNDs.

X-ray photoelectron spectroscopy (XPS) was used to determine the elemental composition, type of bonding, and nature of functional groups. An XPS spectrum of F-CNDs is provided in Figure 4a. The peaks observed at the binding energies (BEs) of 285, 400, and 532 eV indicated the presence of C 1 s, N 1 s, and O 1 s, respectively. Interestingly, the atomic ratio of carbon to other elements was 3:1, and the atomic weight percentages of carbon, nitrogen, and oxygen were 75, 4, and 21%, respectively. Furthermore, the highresolution XPS spectrum ofC1s (Figure 4b) was deconvoluted into five distinct peaks. The binding energy (BE) of the peak at 284.5 eV corresponded to the C=C/C–C bond of

the sp2 and sp3 graphitic structure of F-CNDs [56,57]. The binding energy peak at 285.1 eV corresponded to the pyridinic C–N–C bonds of F-CNDs. The presence of C–OH/C–O–C was confirmed by the peak at 286.1 eV, corresponding to hydroxyl bound to carbon [58]. The peak at 287.0 eV corresponded to C=N/C=O bonds representing pyrrolic nitrogen and carbonyl groups (–C=O) [58], whereas the presence of carboxyl groups (O=C–OH) was confirmed by the peak at 288.5 eV [57]. Figure 4c depicts the XPS spectrum of N 1 s, which exhibited three deconvoluted peaks signifying the presence of pyridinic nitrogen (C–N–C), pyrrolic nitrogen (C–N–H), and graphitic nitrogen (C3–N bonds) with Bes of 399.2, 400.2, and 401.7 eV, respectively [59,60], and showing that F-CND carbon had been doped with nitrogen. Notably, fluorescence results from the ability of excited nitrogendoped carbon to emit light. The chemical type and concentration of nitrogen, carbon structure, and the conditions used for material synthesis can all affect the mechanism of nitrogen-doped carbon fluorescence. However, in most cases, movements of nitrogen electrons to lower energy levels are responsible. The XPS spectrum of O 1 s (Figure 4d) had two deconvoluted peaks at BE 531.5 and 533.1 eV corresponding to C=O/C–OH and C–O–C/O–C=OH, respectively [61]. These findings imply that the surfaces of F-CNDs had –OH, –C–N, and –COOH groups, which provide hydrophilicity and dispersibility in water. Furthermore, ATR-FTIR results were in line with XPS results.

**Figure 2.** (**a**–**c**) HRTEM images of synthesized F-CNDs at different magnifications and (**d**) a particle size distribution histogram.

**Figure 3.** (**a**) Powder XRD pattern, (**b**) Raman spectrum, (**c**) deconvoluted Raman spectrum, and (**d**) ATR-FTIR spectrum of synthesized F-CNDs.

The optical properties of F-CNDs were evaluated using Ultraviolet-visible (UV–vis) absorption and fluorescence spectroscopy. The UV–vis absorption spectrum of F-CNDs (Figure 5a) exhibited two prominent peaks at 217 and 275 corresponding to π–π\* transitions of C–C/C=C and C=C, respectively. In addition, a shoulder was observed at 323, corresponding to the n–π\* transition of C=O or C=N [62]. The inset in Figure 5a demonstrates the dispersion of F-CNDs in water and the difference between exposure to daylight or 365 nm UV light. F-CNDs were dispersed thoroughly in aqueous solvents, and UV exposure resulted in a color change from pale yellow to cyan. This phenomenon was ascribed to the different functional groups on F-CNDs.

F-CNDs exhibited maximum fluorescence at 406 nm when excited at 340 nm (Figure 5b); that is, a Stokes shift of 66 nm occurred. The magnitude of a Stokes shift can significantly impact the practical use of fluorescence. For instance, a significant Stokes shift can improve biological imaging by lowering background noise and increasing the signal-to-noise ratio. However, in some situations, such as in fluorescence resonance energy transfer, a slight spectral overlap between excitation and emission spectra is required to enable energy transfer between fluorescent molecules. The effects of fluorescence excitation wavelengths in the range of 330–420 nm on the emission spectrum of F-CNDs are shown in Figure 5c. Interestingly, the intensity of the emission spectrum increased upon increasing the excitation wavelength from 330 to 340 nm but reduced upon increasing it from 340 to 420 nm, and maximum emission intensity was observed at 340 nm. A normalized excitation-dependent emission spectrum (Figure S1) implies a redshift in the 395 to 495 nm wavelength range. The shift primarily results from electron transfer from the conjugated surface functional

groups narrowing the energy gap. Presumably, if an emitting molecule or fluorophore is in a different environment than the absorbing molecule, a redshift in emission could also occur. In addition, some types of fluorescence, such as two-photon fluorescence, in which two photons of lower energy are simultaneously absorbed, can also cause a redshift. The photostability of F-CNDs was studied by continuously irradiating them with 365 nm UV light at a power of 4 W for 0–120 min (Figure 5d). The intensities of the emission spectra obtained were unchanged without any decay in emission, which confirmed the photostability of F-CNDs. Furthermore, prolonged UV exposure for 120 min caused no color change or precipitate formation (inset of Figure 5d). In addition, the quantum yield of F-CNDs was calculated to be 15.5%. These characteristics of F-CNDs might be due to a wide range of particle sizes, interactions caused by quantum confinement, and the presence of different functional groups.

**Figure 4.** (**a**) XPS-survey spectrum and high-resolution XPS spectra of (**b**) C 1 s, (**c**) N 1 s, and (**d**) O 1 s of synthesized F-CNDs.

In the carbon core of CDs, sp2-conjugated frameworks are typically accompanied by a number of imperfect sp2 domains. These areas will generate or induce surface energy traps that can serve as exciton capture sites, leading to fluorescence associated with the surface defect state. Therefore, surface flaws are responsible for visible light multicolor emissions from CDs. The band gap primarily controls the emission wavelength and is influenced by a wide range of variables, including CD surface chemistry, synthesis techniques, and edge configuration. Due to the epoxy, carboxyl, and hydroxyl groups present in the sp2

clusters, which encompass an extensive spectrum of size distribution, various band gap energies exhibit a variety of emission spectra. Two main types of mechanisms underlie luminescence, namely, quantum confinement in nanometric structures and those involving radiative relaxation of excited states attained by different functional groups within CDs [63]. Furthermore, pyrolytic processing and partial thermal decomposition of precursors cause the formation of intermediate organic fluorophores [64]. Based on our results, we suggest the emission properties of F-CDs are probably due to radiative transitions within or between functional groups on the surfaces of F-CNDs.

**Figure 5.** (**a**) UV-vis absorption spectrum (inset: photographic images of synthesized F-CNDs in aqueous solution under daylight (**left**) and 365 nm UV light (**right**)); (**b**) fluorescence excitation and emission spectra, and (**c**) fluorescence excitation-dependent emission spectra of synthesized F-CNDs. (**d**) Fluorescence emission spectra of synthesized F-CNDs before and after continuous irradiation with 365 nm UV light (inset: photographic images of synthesized F-CNDs in aqueous suspension under 365 nm UV light before (0 min) and after (120 min) continuous irradiation with 365 nm UV).

F-CNDs emit controllable fluorescence, have appropriate quantum yields, high water dispersibility, low cytotoxicity, and excellent biocompatibility, and do not exhibit photobleaching. The produced F-CNDs were used for cellular imaging without modification. MTT cell viability test results for HCT-116 cells (a human colon cancer cell line) at F-CND concentrations of 0 to 200 μg mL–1 are shown in Figure S2. The bar chart provides a comparison between the viabilities of F-CND treated and untreated cells (controls) and shows a slight decrease (from 100 to 97%) in cell viabilities with increasing concentration of F-CNDs from 0 to 200 μg mL–1. This observation indicated good compatibility and low cytotoxicity

of F-CNDs with a human colon cancer cell line, and cytotoxicity does not lead to cell death even at higher concentrations of 200 μg mL–1, which is an essential property required for F-CNDs to make them suitable for bioimaging of cells. To comprehend the dynamics, one must first understand how F-CNDs become internalized within cells, tissues, or cellular cytoskeleton components. Actin filaments, Microtubules, and intermediate filaments are intracellular components that actively collaborate with cancer cells. Confocal microscopy was used to investigate the bioimaging characteristics of F-CNDs in human colon cancer cells. Figure 6 contains confocal microscopy photographs of HCT-116 cells, treated or not with F-CNDs, taken using different wavelength filters, viz. 405 (blue), 488 (green), and 555 nm (red) after exposure to bright field illumination for 12 or 24 h. No emission was observed from untreated HCT-116 cells, whereas fluorescence was observed from human colon cancer cells treated with F-CNDs when 405, 488, or 555 nm filters were used, which produced blue, green, and red emissions, respectively. The overlapping image was multicolored (Figure 6), indicating excitation wavelength-dependent emission characteristics. Upon increasing the exposure time from 12 h to 24 h, enhancement in the intensity of fluorescence is well observed from the image. It has been well established that F-CNDs are easily internalized and uniformly distributed in human colon cancer cells. Therefore, these results show that F-CNDs are candidate fluorescent nanoprobes for imaging human colon cancer cells.

**Figure 6.** Confocal microscopy fluorescence images of human colon cancer cells treated with or without F-CNDs and the synthesized F-CNDs treated for 12 and 24 h with the concentration of 100 μg mL−<sup>1</sup> using different excitation filters 405, 488, and 555 nm (blue, green, and red, respectively) as well as bright-field illumination.

#### **3. Materials and Methods**

#### *3.1. Materials*

Cashew nut skin waste was collected from Tamil Nadu, India. Aqueous ammonia (NH4OH, 25%) was purchased from Sigma-Aldrich, Republic of Korea. Phosphate buffered saline (PBS), N-(2-hydroxyethyl)piperazine-N'-(2-ethane sulfonic acid) (HEPES), p-formaldehyde, quinine sulfate, and dimethyl sulfoxide (DMSO) were purchased from Sigma-Aldrich, Republic of Korea. 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium

bromide (MTT) was purchased from Generay Biotech, Shanghai, China. HCT-116 human colon cancer cells were purchased from ATCC, CCL-247, Manassas, VA, USA. All the chemicals were used as purchased and distilled water was used throughout this study.

#### *3.2. The Synthesis of Functionalized Carbon Nanodots*

F-CNDs were synthesized using washed, dried, and ground cashew nut skins. The whitish-brown powder obtained was added to 50 mL of water with 1 mL of 25% ammonium hydroxide solution and placed in an autoclave at 200 ◦C for 24 h. Large carbon particles were eliminated via filtration, and the filtrate was passed through a mixed cellulose ester membrane filter with a pore size of 0.22 μm, frozen in liquid nitrogen, and dried at below −80 ◦C in a freeze dryer. The F-CNDs obtained were used in subsequent experiments. Scheme 1 shows the synthesis procedure of F-CNDs from cashew nut skin waste using the hydrothermal-carbonization.

**Scheme 1.** Hydrothermal synthesis of functionalized carbon nanodots from cashew nut skin waste.

#### **4. Conclusions**

Using a single-step method, cashew nut skin waste was used to synthesize F-CNDs using a simple hydrothermal route at a very low temperature without any further modifications that were quite economical. The formations of F-CNDs were considered to be due to the dehydration, polymerization, and carbonization of hydrolyzable tannins, and phenolic acids present in the testa of cashew nuts. F-CNDs exhibited a graphitic structure at the core with few surface defects as determined by XRD and Raman Spectroscopy. F-CNDs had a mean particle size of 2.5 nm and were composed of carbon, nitrogen, and oxygen decorated with functional groups (C=O, –OH, –NH2, and –COOH), as determined by XPS and ATR-FTIR, which conferred F-CNDs with significant hydrophilicity and dispersibility. F-CNDs had excellent fluorescent properties and exhibited maximum emission at 406 nm when excited at 340 nm due to radiative transitions within or between functional groups present on the surfaces of F-CNDs. F-CNDs were photostable, had a quantum yield of 15.5%, and at concentrations of 0–200 μg mL−<sup>1</sup> returned MTT viability greater than 95% for HCT-116 cells. F-CNDs thus proved to have remarkable biocompatibility and low cytotoxicity with the cancer cell line. Confocal microscopy of human colon cancer cells treated with or without F-CNDs revealed blue, green, and red emissions when exposed to 405, 488, and 555 nm light, respectively, in addition to a bright field. After increasing the time of exposure from 12 h to 24 h, significant enhancement in the intensity of fluorescence was observed. Our results show nano-sized, cashew-nut-skin-derived F-CNDs have a graphitized core structure and are surface functionalized by organic moieties. They are suitable nanoprobes for bioimaging, drug delivery, and cell labeling. In the near future, a material that is safe for the delivery of anticancer drugs could be developed using the successful integration of F-CNDs with anticancer drugs.

**Supplementary Materials:** The following are available online at https://www.mdpi.com/article/10 .3390/catal13030547/s1. Instrumentation methods, quantum yield measurements, photobleaching measurements, cell culture, cell viability assay, and microscopy results. Figure S1. Fluorescence excitation-dependent emission normalized-spectra of synthesized F-CNDs; Figure S2. Cell viability MTT assay results. The bar chart provides a comparison of the viabilities of F-CND treated cells and untreated controls.

**Author Contributions:** Investigation and writing of the original draft, S.C.K.; visualization, reviewing the original draft, and editing, S.P.; conceptualization, data curation, formal analysis, investigation, and writing the original draft, R.A.; formal analysis and investigation T.N.J.I.E.; investigation and visualization, A.K.S.; investigation and validation, M.A.; formal analysis and visualization, S.S.; project administration and supervision, Y.R.L. The authors contributed equally to this work. All authors have read and agreed to the published version of the manuscript.

**Funding:** This research received no external funding.

**Data Availability Statement:** Not applicable.

**Conflicts of Interest:** The authors declare no conflict of interest.

#### **References**


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